Sulfamide linkers for use in bioconjugates

ABSTRACT

The invention relates to a novel linker for use in bioconjugates such as antibody-drug-conjugates. The linker according to the invention is represented by formula: (I) wherein: —BM is a branching moiety; —E is a capping group; —SG is a sulfamide group; —b, c, d, e, g, i, k, l are independently 0 or 1; —f is an integer in the range of 1 to 10; —Sp 1 , Sp 2 , Sp 3 , Sp 4 , Sp 5  and Sp 6  are a spacer moieties; —Z 1  and Z 2  are connecting groups. The linker according to the invention is useful in the preparation of linker-conjugates and bioconjugates, and can be used for (a) improving conjugation efficiency in the preparation of the bioconjugate, (b) reducing aggregation during the preparation of the bioconjugate and/or of the bioconjugate, (c) increasing stability of the bioconjugate, and/or (d) increasing therapeutic index of the bioconjugate.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. national stage application ofPCT/EP2017/052719, filed Feb. 8, 2017, which claims the benefit of andpriority to European Patent Application No. 16154739.3, filed Feb. 8,2016, the entire contents of which are incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-WEB and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 7, 2018, isnamed 069818-4060 2018-08-07 _Sequence Listing.txt and is 29 KB.

FIELD OF THE INVENTION

The present invention is in the field of bioconjugation. The inventionrelates to novel sulfamide linkers and conjugates thereof, and tomethods for the preparation thereof. More particularly, the inventionrelates to linkers comprising an acylsulfamide group and/or a carbamoylsulfamide group and to conjugates comprising said linkers. The inventionfurther relates to a process for the preparation of bioconjugatescomprising a linker, the linker comprising an acylsulfamide group and/ora carbamoyl sulfamide group.

BACKGROUND

Bioconjugation is the process of linking two or more molecules, of whichat least one is a biomolecule. The biomolecule(s) may also be referredto as “biomolecule(s) of interest”, the other molecule(s) may also bereferred to as “target molecule” or “molecule of interest”. Typicallythe biomolecule of interest (BOI) will consist of a protein (orpeptide), a glycan, a nucleic acid (or oligonucleotide), a lipid, ahormone or a natural drug (or fragments or combinations thereof). Theother molecule of interest (MOI) may also be a biomolecule, henceleading to the formation of homo- or heterodimers (or higher oligomers),or the other molecule may possess specific features that are impartedonto the biomolecule of interest by the conjugation process. Forexample, the modulation of protein structure and function by covalentmodification with a chemical probe for detection and/or isolation hasevolved as a powerful tool in proteome-based research and biomedicalapplications. Fluorescent or affinity tagging of proteins is key tostudying the trafficking of proteins in their native habitat. Vaccinesbased on protein-carbohydrate conjugates have gained prominence in thefight against HIV, cancer, malaria and pathogenic bacteria, whereascarbohydrates immobilized on microarrays are instrumental in elucidationof the glycome. Synthetic DNA and RNA oligonucleotides (ONs) require theintroduction of a suitable functionality for diagnostic and therapeuticapplications, such as microarray technology, antisense andgene-silencing therapies, nanotechnology and various materials sciencesapplications. For example, attachment of a cell-penetrating ligand isthe most commonly applied strategy to tackle the low internalizationrate of ONs encountered during oligonucleotide-based therapeutics(antisense, siRNA). Similarly, the preparation of oligonucleotide-basedmicroarrays requires the selective immobilization of ONs on a suitablesolid surface, e.g. glass.

There are numerous examples of chemical reactions suitable to covalentlylink two (or more) molecular structures. However, labelling ofbiomolecules poses high restrictions on the reaction conditions that canbe applied (solvent, concentration, temperature), while the desire ofchemoselective labelling limits the choice of reactive groups. Forobvious reasons, biological systems generally flourish best in anaqueous environment meaning that reagents for bioconjugation should besuitable for application in aqueous systems. In general, two strategicconcepts can be recognized in the field of bioconjugation technology:(a) conjugation based on a functional group already present in thebiomolecule of interest, such as for example a thiol, an amine, analcohol or a hydroxyphenol unit or (b) a two-stage process involvingengineering of one (or more) unique reactive groups into a BOI prior tothe actual conjugation process.

The first approach typically involves a reactive amino acid side-chainin a protein (e.g. cysteine, lysine, serine and tyrosine), or afunctional group in a glycan (e.g. amine, aldehyde) or nucleic acid(e.g. purine or pyrimidine functionality or alcohol). As summarizedinter alia in G. T. Hermanson, “Bioconjugate Techniques”, Elsevier,3^(rd) Ed. 2013, incorporated by reference, a large number of reactivefunctional groups have become available over the years forchemoselective targeting of one of these functional groups, such asmaleimide, haloacetamide, activated ester, activated carbonate, sulfonylhalide, activated thiol derivative, alkene, alkyne, allenamide and more,each of which requiring its own specific conditions for conjugation (pH,concentration, stoichiometry, light, etc.). Most prominently,cysteine-maleimide conjugation stands out for protein conjugation byvirtue of its high reaction rate and chemoselectivity. However, when nocysteine is available for conjugation, as in many proteins and certainlyin other biomolecules, other methods are often required, each sufferingfrom its own shortcomings.

An elegant and broadly applicable solution for bioconjugation involvesthe two-stage approach. Although more laborious, two-stage conjugationvia engineered functionality typically leads to higher selectivity(site-specificity) than conjugation on a natural functionality. Besidesthat, full stability can be achieved by proper choice of construct,which can be an important shortcoming of one stage conjugation on nativefunctionality, in particular for cysteine-maleimide conjugation. Typicalexamples of a functional group that may be imparted onto the BOI include(strained) alkyne, (strained) alkene, norbornene, tetrazine, azide,phosphine, nitrile oxide, nitrone, nitrile imine, diazo compound,carbonyl compound, (O-alkyl)hydroxylamine and hydrazine, which may beachieved by either chemical or molecular biology approach. Each of theabove functional groups is known to have at least one reaction partner,in many cases involving complete mutual reactivity. For example,cyclooctynes react selectively and exclusively with 1,3-dipoles,strained alkenes with tetrazines and phosphines with azides, leading tofully stable covalent bonds. However, some of the above functionalgroups have the disadvantage of being highly lipophilic, which maycompromise conjugation efficiency, in particular in combination with alipophilic molecule of interest (see below).

The final linking unit between the biomolecule and the other molecule ofinterest should preferentially also be fully compatible with an aqueousenvironment in terms of solubility, stability and biocompatibility. Forexample, a highly lipophilic linker may lead to aggregation (duringand/or after conjugation), which may significantly increase reactiontimes and/or reduce conjugation yields, in particular when the MOI isalso of hydrophobic nature. Similarly, highly lipophilic linker-MOIcombination may lead to unspecific binding to surfaces or specifichydrophobic patches on the same or other biomolecules. If the linker issusceptible to aqueous hydrolysis or other water-induced cleavagereactions, the components comprising the original bioconjugate separateby diffusion. For example, certain ester moieties are not suitable dueto saponification while 3-hydroxycarbonyl or γ-dicarbonyl compoundscould lead to retro-aldol or retro-Michael reaction, respectively.Finally, the linker should be inert to functionalities present in thebioconjugate or any other functionality that may be encountered duringapplication of the bioconjugate, which excludes, amongst others, the useof linkers featuring for example a ketone or aldehyde moiety (may leadto imine formation), an α,β-unsaturated carbonyl compound (Michaeladdition), thioesters or other activated esters (amide bond formation).

Compounds made of linear oligomers of ethylene glycol, so-calledpolyethylene glycol (PEG) linkers, enjoy particular popularity nowadaysin biomolecular conjugation processes. PEG linkers are highly watersoluble, non-toxic, non-antigenic, and lead to negligible or noaggregation. For this reason, a large variety of linear, bifunctionalPEG linkers are commercially available from various sources, which canbe selectively modified at either end with a (bio)molecule of interest.PEG linkers are the product of a polymerization process of ethyleneoxide and are therefore typically obtained as stochastic mixtures ofchain length, which can be partly resolved into PEG constructs with anaverage weight distribution centered around 1, 2, 4 kDa or more (up to60 kDa). Homogeneous, discrete PEGs (dPEGs) are also known withmolecular weights up to 4 kDa and branched versions thereof go up to 15kDa. Interestingly, the PEG unit itself imparts particularcharacteristics onto a biomolecule. In particular, protein PEGylationmay lead to prolonged residence in vivo, decreased degradation bymetabolic enzymes and a reduction or elimination of proteinimmunogenicity. Several PEGylated proteins have been FDA-approved andare currently on the market.

By virtue of their polarity, PEG linkers are suitable for bioconjugationof small and/or water-soluble moieties under aqueous conditions.However, in case of conjugation of hydrophobic, water-insolublemolecules of interest, the polarity of a PEG unit may be insufficient tooffset hydrophobicity, leading to significantly reduced reaction rates,lower yields and induced aggregation issues. In such case, lengthy PEGlinkers and/or significant amounts of organic co-solvents may berequired to solubilize the reagents. For example, in the field ofantibody-drug conjugates, the controlled attachment of a distinct numberof toxic payloads to a monoclonal antibody is key, with a payloadtypically selected from the group of auristatins, maytansinoids,duocarmycins, enediynes or pyrrolobenzodiazepines (PBDs), with manyothers are underway. With the exception of auristatin F, all toxicpayloads are poorly soluble or water-insoluble, which necessitatesorganic co-solvents to achieve successful conjugation, such as 25%N,N-dimethylacetamide (DMA) or dimethylformamide (DMF) or up to 50%propylene glycol (PG). In case of hydrophobic payloads, despite the useof aforementioned co-solvents, large stoichiometries of reagents may berequired during conjugation while efficiency and yield may besignificantly compromised due to aggregation (in process or afterproduct isolation), as for example described by Senter et al. in Nat.Biotechn. 2014, 24, 1256-1263, incorporated by reference. The use oflong PEG spacers (12 units or more) may partially enhance solubilityand/or conjugation efficiency, but it has been shown that long PEGspacers may lead to more rapid in vivo clearance, and hence negativelyinfluence the pharmacokinetic profile of the ADC.

Using conventional linkers (e.g. PEG), effective conjugation is oftenhampered by the relatively low solubility of the linker-conjugate inaqueous media, especially when a relative water-insoluble or hydrophobictarget molecule is used. In their quest for a short, polar spacer thatenables fast and efficient conjugation of hydrophobic moieties, theinventors have developed the sulfamide linker, which was found toimprove the solubility of the linker-conjugate, which in turnsignificantly improves the efficiency of the conjugation and reducesboth in process and product aggregation. This is disclosed in patentapplication PCT/NL2015/050697 (WO 2016/053107), which is incorporatedherein in its entirety.

Further linkers are known in the art, and disclosed in e.g. WO2008/070291, incorporated by reference. WO 2008/070291 discloses alinker for the coupling of targeting agents to anchoring components. Thelinker contains hydrophilic regions represented by polyethylene glycol(PEG) and an extension lacking chiral centers that is coupled to atargeting agent.

WO 01/88535, incorporated by reference, discloses a linker system forsurfaces for bioconjugation, in particular a linker system having anovel hydrophilic spacer group. The hydrophilic atoms or groups for usein the linker system are selected from the group consisting of O, NH,C═O (keto group), O—C═O (ester group) and CR³R⁴, wherein R³ and R⁴ areindependently selected from the group consisting of H, OH, C₁-C₄ alkoxyand C₁-C₄ acyloxy.

WO 2014/100762, incorporated by reference, describes compounds with ahydrophilic self-immolative linker, which is cleavable under appropriateconditions and incorporates a hydrophilic group to provide bettersolubility of the compound. The compounds comprise a drug moiety, atargeting moiety capable of targeting a selected cell population, and alinker which contains an acyl unit, an optional spacer unit forproviding distance between the drug moiety and the targeting moiety, apeptide linker which can be cleavable under appropriate conditions, ahydrophilic self-immolative linker, and an optional secondself-immolative spacer or cyclization self-elimination linker. Thehydrophilic self-immolative linker is e.g. a benzyloxycarbonyl group.

SUMMARY OF THE INVENTION

The present invention relates to an improved linker that can be used inthe field of bioconjugation. The sulfamide linker that has beendeveloped by the inventors has been put to practice in the followingaspects of the invention.

In a first aspect, the invention concerns a linker-conjugate as definedherein, comprising the linker according to the invention, as well as theuse of said linker-conjugate for preparing a bioconjugate according tothe invention. In a second aspect, the invention concerns alinker-construct as defined herein, comprising the linker according tothe invention, as well as the use of said linker-construct for preparinga linker-conjugate according to the invention and a process forpreparing the linker-conjugate according to the invention using saidlinker-construct. In a third aspect, the invention concerns abioconjugate as defined herein, comprising the linker according to theinvention. In a fourth aspect, the invention concerns a process forpreparing the bioconjugate according to the invention and thebioconjugate obtainable thereby. In a fifth aspect, the inventionconcerns the use of the linker according to the invention inbioconjugation for improving conjugation efficiency, reducingaggregation, increasing stability and increasing therapeutic index. In asixth aspect, the invention concerns the medical use of the bioconjugateaccording to the invention, in particular for the treatment of cancer.

The linker-conjugate according to the invention as a compound wherein atarget molecule D is covalently connected to a reactive group Q¹, via alinker, wherein the linker is represented by formula:

wherein:

-   -   BM is a branching moiety;    -   E is a capping group;    -   SG is a sulfamide group according to formula (1);    -   b is independently 0 or 1;    -   c is 0 or 1;    -   d is 0 or 1;    -   e is 0 or 1;    -   f is an integer in the range of 1 to 10;    -   g is 0 or 1;    -   i is 0 or 1;    -   k is 0 or 1;    -   l is 0 or 1;    -   Sp¹ is a spacer moiety;    -   Sp² is a spacer moiety;    -   Sp³ is a spacer moiety;    -   Sp⁴ is a spacer moiety;    -   Sp⁵ is a spacer moiety;    -   Sp⁶ is a spacer moiety;    -   Z¹ is a connecting group;    -   Z² is a connecting group,

wherein one of the bonds labelled with * is connected to reactive groupQ¹ and one of the bonds labelled with * is connected to target moleculeD, and wherein the sulfamide group SG is represented by formula (1):

wherein

-   -   a is 0 or 1; and    -   R¹ is selected from the group consisting of hydrogen, C₁-C₂₄        alkyl groups, C₃-C₂₄ cycloalkyl groups, C₂-C₂₄ (hetero)aryl        groups, C₃-C₂₄ alkyl(hetero)aryl groups and C₃-C₂₄        (hetero)arylalkyl groups, wherein the C₁-C₂₄ alkyl groups,        C₃-C₂₄ cycloalkyl groups, C₂-C₂₄ (hetero)aryl groups, C₃-C₂₄        alkyl(hetero)aryl groups and C₃-C₂₄ (hetero)arylalkyl groups are        optionally substituted and optionally interrupted by one or more        heteroatoms selected from O, S and NR³ wherein R³ is        independently selected from the group consisting of hydrogen and        C₁-C₄ alkyl groups, or R¹ is a further target molecule D,        wherein the target molecule is optionally connected to N via a        spacer moiety,

and wherein one of the bonds labelled with * is connected to thebranching moiety, optionally via spacer Sp⁵, and the other bond labelledwith * to a capping group E, optionally via spacer Sp⁶. The bioconjugateaccording to the invention is a compound wherein a target molecule D iscovalently connected to a biomolecule B, via a linker, wherein thelinker is represented by formula:

wherein:

-   -   BM is a branching moiety;    -   E is a capping group;    -   SG is a sulfamide group according to formula (1);    -   b is independently 0 or 1;    -   c is 0 or 1;    -   d is 0 or 1;    -   e is 0 or 1;    -   f is an integer in the range of 1 to 10;    -   g is 0 or 1;    -   i is 0 or 1;    -   k is 0 or 1;    -   l is 0 or 1;    -   Sp¹ is a spacer moiety;    -   Sp² is a spacer moiety;    -   Sp³ is a spacer moiety;    -   Sp⁴ is a spacer moiety;    -   Sp⁵ is a spacer moiety;    -   Sp⁶ is a spacer moiety;    -   Z¹ is a connecting group;    -   Z² is a connecting group,

wherein one of the bonds labelled with * is connected to biomolecule Band one of the bonds labelled with * is connected to target molecule D,and wherein the sulfamide group SG is represented by formula (1):

wherein

-   -   a is 0 or 1; and    -   R¹ is selected from the group consisting of hydrogen, C₁-C₂₄        alkyl groups, C₃-C₂₄ cycloalkyl groups, C₂-C₂₄ (hetero)aryl        groups, C₃-C₂₄ alkyl(hetero)aryl groups and C₃-C₂₄        (hetero)arylalkyl groups, wherein the C₁-C₂₄ alkyl groups,        C₃-C₂₄ cycloalkyl groups, C₂-C₂₄ (hetero)aryl groups, C₃-C₂₄        alkyl(hetero)aryl groups and C₃-C₂₄ (hetero)arylalkyl groups are        optionally substituted and optionally interrupted by one or more        heteroatoms selected from O, S and NR³ wherein R³ is        independently selected from the group consisting of hydrogen and        C₁-C₄ alkyl groups, or R¹ is a further target molecule D,        wherein the target molecule is optionally connected to N via a        spacer moiety,

and wherein one of the bonds labelled with * is connected to thebranching moiety, optionally via spacer Sp⁵, and the other bond labelledwith * to a capping group E, optionally via spacer Sp⁶. The inventionfurther concerns a process for the preparation of a bioconjugate,comprising reacting a reactive group Q¹ of the compound according to anyone of claims 1-6 with a functional group F¹ of a biomolecule (B).

The invention further concerns the use of the linker-conjugate accordingto the invention for the preparation of a bioconjugate.

The invention further concerns the use of a linker represented byformula:

wherein:

-   -   BM is a branching moiety;    -   E is a capping group;    -   SG is a sulfamide group according to formula (1);    -   b is independently 0 or 1;    -   c is 0 or 1;    -   d is 0 or 1;    -   e is 0 or 1;    -   f is an integer in the range of 1 to 10;    -   g is 0 or 1;    -   i is 0 or 1;    -   k is 0 or 1;    -   l is 0 or 1;    -   Sp¹ is a spacer moiety;    -   Sp² is a spacer moiety;    -   Sp³ is a spacer moiety;    -   Sp⁴ is a spacer moiety;    -   Sp⁵ is a spacer moiety;    -   Sp⁶ is a spacer moiety;    -   Z¹ is a connecting group;    -   Z² is a connecting group,

wherein one of the bonds labelled with * is connected to biomolecule Band one of the bonds labelled with * is connected to target molecule D,and wherein the sulfamide group SG is represented by formula (1):

wherein

-   -   a is 0 or 1; and    -   R¹ is selected from the group consisting of hydrogen, C₁-C₂₄        alkyl groups, C₃-C₂₄ cycloalkyl groups, C₂-C₂₄ (hetero)aryl        groups, C₃-C₂₄ alkyl(hetero)aryl groups and C₃-C₂₄        (hetero)arylalkyl groups, wherein the C₁-C₂₄ alkyl groups,        C₃-C₂₄ cycloalkyl groups, C₂-C₂₄ (hetero)aryl groups, C₃-C₂₄        alkyl(hetero)aryl groups and C₃-C₂₄ (hetero)arylalkyl groups are        optionally substituted and optionally interrupted by one or more        heteroatoms selected from O, S and NR³ wherein R³ is        independently selected from the group consisting of hydrogen and        C₁-C₄ alkyl groups, or R¹ is a further target molecule D,        wherein the target molecule is optionally connected to N via a        spacer moiety,

and wherein one of the bonds labelled with * is connected to thebranching moiety, optionally via spacer Sp⁵, and the other bond labelledwith * to a capping group E, optionally via spacer Sp⁶, in abioconjugate for:

-   -   (a) improving conjugation efficiency in the preparation of the        bioconjugate,    -   (b) reducing aggregation during the preparation of the        bioconjugate and/or of the bioconjugate,    -   (c) increasing stability of the bioconjugate, and/or    -   (d) increasing therapeutic index of the bioconjugate.

The invention further concerns the bioconjugate according to theinvention for use in the treatment of a subject in need thereof.

DESCRIPTION OF DRAWINGS

FIG. 1 describes the general concept of conjugation of biomolecules: abiomolecule of interest (BOI) containing one or more functional groupsF¹ is incubated with (excess of) a target molecule D (also referred toas molecule of interest or MOI) covalently attached to a reactive groupQ¹ via a specific linker. In the process of bioconjugation, a chemicalreaction between F¹ and Q¹ takes place, thereby forming a bioconjugatecomprising a covalent connection between the BOI and the MOI. The BOImay e.g. be a peptide/protein, a glycan or a nucleic acid.

FIG. 2 shows several structures of derivatives of UDP sugars ofgalactosamine, which may be modified with e.g. a 3-mercaptopropionylgroup (11a), an azidoacetyl group (11b), or an azidodifluoroacetyl group(11c) at the 2-position, or with an azido group at the 6-position ofN-acetyl galactosamine (11d).

FIG. 3 schematically displays how any of the UDP-sugars 11a-d may beattached to a glycoprotein comprising a GlcNAc moiety 12 (e.g. amonoclonal antibody the glycan of which is trimmed by anendoglycosidase) under the action of a galactosyltransferase mutant or aGalNAc-transferase, thereby generating a 3-glycosidic 1-4 linkagebetween a GalNAc derivative and GlcNAc (compounds 13a-d, respectively).

FIG. 4 shows how a modified antibody 13a-d may undergo a bioconjugationprocess by means of nucleophilic addition to maleimide (as for3-mercaptopropionyl-galactosamine-modified 13a leading to thioetherconjugate 14, or for conjugation to a engineered cysteine residueleading to thioether conjugate 17) or upon strain-promoted cycloadditionwith a cyclooctyne reagent (as for 13b, 13c or 13d leading to triazoles15a, 15b or 16, respectively).

FIG. 5 depicts the general structure of the linker-construct (FIG. 5A),the linker-conjugate (FIG. 5B) and the bioconjugate (FIG. 5C) accordingto the invention. Herein, B refers to biomolecule, D refers to targetmolecule, SG refers to sulfamide group, E refers to capping moiety, BMrefers to branching moiety and Q¹ and Q² are reactive groups.

FIG. 6 shows a representative set of functional groups (F¹) in abiomolecule, either naturally present or introduced by engineering,which upon reaction with reactive group Q¹ lead to connecting group Z³.Functional group F¹ may also be artificially introduced (engineered)into a biomolecule at any position of choice. The same functional groupsand reactive moieties are suitably used as F² and Q², respectively.Connecting group Z³ may also be the result of a reaction between F² andQ².

DETAILED DESCRIPTION

The present invention relates to an improved linker that can be used inthe field of bioconjugation. The sulfamide linker that has beendeveloped by the inventors has been put to practice in the followingaspects of the invention.

In a first aspect, the invention concerns a linker-conjugate as definedherein, comprising the linker according to the invention, as well as theuse of said linker-conjugate for preparing a bioconjugate according tothe invention. In a second aspect, the invention concerns alinker-construct as defined herein, comprising the linker according tothe invention, as well as the use of said linker-construct for preparinga linker-conjugate according to the invention and a process forpreparing the linker-conjugate according to the invention using saidlinker-construct. In a third aspect, the invention concerns abioconjugate as defined herein, comprising the linker according to theinvention. In a fourth aspect, the invention concerns a process forpreparing the bioconjugate according to the invention and thebioconjugate obtainable thereby. In a fifth aspect, the inventionconcerns the use of the linker according to the invention inbioconjugation for improving conjugation efficiency, reducingaggregation, increasing stability and increasing therapeutic index. In asixth aspect, the invention concerns the medical use of the bioconjugateaccording to the invention, in particular for the treatment of cancer.

Key to all of the above aspects to the invention is the linker accordingto the invention, which comprises a branching moiety BM in the mainchain of the linker and a sulfamide group SG in the side chain of thelinker. Herein, “main chain” refers to the chain of atoms that connectstarget molecule D (or Q¹) with biomolecule B (or Q²). The branchingmoiety is located in the main chain. The “side chain” refers to thechain of atoms that connects branching moiety BM to capping group E,also referred to as the “branch”. The linker according to the inventionis further defined below. The inventors surprisingly found that using asulfamide group within the side chain (branch), in the configurationaccording to the present invention, provides many advantages whenpreparing and using bioconjugates such as antibody-drug-conjugates.

First and foremost, all the advantages associated with sulfamide groupsembedded in the chain of a linker, i.e. biomolecule B or reactive groupQ¹ is connected to one end of the sulfamide group and target molecule Dor reactive group Q² is connected to the other end of the sulfamidegroup, also hold for the present configuration, wherein the sulfamidemoiety is not located in the chain between target molecule andbiomolecule, but located in a branch of the chain. The advantages interms of conjugation efficiency and reduced aggregation are described inPCT/NL2015/020697, which is incorporated herein by reference in itsentirety. Using conventional linkers (e.g. PEG), effective conjugationis often hampered by the relatively low solubility of thelinker-conjugate in aqueous media, especially when a relativewater-insoluble or hydrophobic target molecule is used. In their questfor a short, polar spacer that enables fast and efficient conjugation ofhydrophobic moieties, the inventors have developed the sulfamide linker,which was found to improve the solubility of the linker-conjugate, whichin turn significantly improves the efficacy efficiency of theconjugation and reduces aggregation both in process and product. Theinventors found that the sulfamide linkers according to the presentinvention are capable of (a) improving conjugation efficiency in thepreparation of the bioconjugate, (b) reducing aggregation during thepreparation of the bioconjugate and/or of the bioconjugate, (c)increasing stability of the bioconjugate, and/or (d) increasingtherapeutic index of the bioconjugate.

The inventors further found that the bioconjugates according to thepresent invention, thus comprising the branched sulfamide linkeraccording to the present invention, exhibited a greater therapeuticefficacy compared to the same bioconjugate, i.e. the same biomolecule,the same active substance (drug) and the same biomolecule drug ratio,containing a linker not according to the present invention. That thestructure of the linker could have an effect on the therapeutic efficacyof a bioconjugate, such as an antibody-drug-conjugate, could not beenvisioned based on the current knowledge. In the field, linkers areconsidered inert when it comes to treatment and are solely present as aconsequence of the preparation of the bioconjugate. That a specificstructural element within a linker has an effect on the therapeuticefficacy is unprecedented in the art and a breakthrough discovery in thefield of bioconjugates, in particular antibody-drug-conjugates. Thebioconjugates according to the invention are thus more therapeuticallyeffective as the same bioconjugates, i.e. the same biomolecule, the sameactive substance (drug) and the same biomolecule drug ratio, containinga different linker. This finding has dramatic implications on thetreatment of subjects with the bioconjugate according to the invention,as treatment doses may be lowered and as a consequence potential,unwanted, side-effects are reduced. Alternatively, as a result of theincreased tolerability of the bioconjugate (i.e. increased stability andlow aggregation potential) according to the invention, treatment dosesmight be increased without the potential increase in unwantedside-effects.

Definitions

The verb “to comprise”, and its conjugations, as used in thisdescription and in the claims is used in its non-limiting sense to meanthat items following the word are included, but items not specificallymentioned are not excluded. In addition, reference to an element by theindefinite article “a” or “an” does not exclude the possibility thatmore than one of the element is present, unless the context clearlyrequires that there is one and only one of the elements. The indefinitearticle “a” or “an” thus usually means “at least one”. The compoundsdisclosed in this description and in the claims may comprise one or moreasymmetric centres, and different diastereomers and/or enantiomers mayexist of the compounds. The description of any compound in thisdescription and in the claims is meant to include all diastereomers, andmixtures thereof, unless stated otherwise. In addition, the descriptionof any compound in this description and in the claims is meant toinclude both the individual enantiomers, as well as any mixture, racemicor otherwise, of the enantiomers, unless stated otherwise. When thestructure of a compound is depicted as a specific enantiomer, it is tobe understood that the invention of the present application is notlimited to that specific enantiomer.

The compounds may occur in different tautomeric forms. The compoundsaccording to the invention are meant to include all tautomeric forms,unless stated otherwise. When the structure of a compound is depicted asa specific tautomer, it is to be understood that the invention of thepresent application is not limited to that specific tautomer.

The compounds disclosed in this description and in the claims mayfurther exist as exo and endo diastereoisomers. Unless stated otherwise,the description of any compound in the description and in the claims ismeant to include both the individual exo and the individual endodiastereoisomers of a compound, as well as mixtures thereof. When thestructure of a compound is depicted as a specific endo or exodiastereomer, it is to be understood that the invention of the presentapplication is not limited to that specific endo or exo diastereomer.

Furthermore, the compounds disclosed in this description and in theclaims may exist as cis and trans isomers. Unless stated otherwise, thedescription of any compound in the description and in the claims ismeant to include both the individual cis and the individual trans isomerof a compound, as well as mixtures thereof. As an example, when thestructure of a compound is depicted as a cis isomer, it is to beunderstood that the corresponding trans isomer or mixtures of the cisand trans isomer are not excluded from the invention of the presentapplication. When the structure of a compound is depicted as a specificcis or trans isomer, it is to be understood that the invention of thepresent application is not limited to that specific cis or trans isomer.

The compounds according to the invention may exist in salt form, whichare also covered by the present invention. The salt is typically apharmaceutically acceptable salt, containing a pharmaceuticallyacceptable anion. The term “salt thereof” means a compound formed whenan acidic proton, typically a proton of an acid, is replaced by acation, such as a metal cation or an organic cation and the like. Whereapplicable, the salt is a pharmaceutically acceptable salt, althoughthis is not required for salts that are not intended for administrationto a patient. For example, in a salt of a compound the compound may beprotonated by an inorganic or organic acid to form a cation, with theconjugate base of the inorganic or organic acid as the anionic componentof the salt.

The term “pharmaceutically accepted” salt means a salt that isacceptable for administration to a patient, such as a mammal (salts withcounter ions having acceptable mammalian safety for a given dosageregime). Such salts may be derived from pharmaceutically acceptableinorganic or organic bases and from pharmaceutically acceptableinorganic or organic acids. “Pharmaceutically acceptable salt” refers topharmaceutically acceptable salts of a compound, which salts are derivedfrom a variety of organic and inorganic counter ions known in the artand include, for example, sodium, potassium, calcium, magnesium,ammonium, tetraalkylammonium, etc., and when the molecule contains abasic functionality, salts of organic or inorganic acids, such ashydrochloride, hydrobromide, formate, tartrate, besylate, mesylate,acetate, maleate, oxalate, etc.

Unsubstituted alkyl groups have the general formula C_(n)H_(2n+1) andmay be linear or branched. Optionally, the alkyl groups are substitutedby one or more substituents further specified in this document. Examplesof alkyl groups include methyl, ethyl, propyl, 2-propyl, t-butyl,1-hexyl, 1-dodecyl, etc.

A cycloalkyl group is a cyclic alkyl group. Unsubstituted cycloalkylgroups comprise at least three carbon atoms and have the general formulaC_(n)H_(2n-1). Optionally, the cycloalkyl groups are substituted by oneor more substituents further specified in this document. Examples ofcycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl andcyclohexyl.

An alkenyl group comprises one or more carbon-carbon double bonds, andmay be linear or branched. Unsubstituted alkenyl groups comprising oneC—C double bond have the general formula C_(n)H_(2n-1). Unsubstitutedalkenyl groups comprising two C—C double bonds have the general formulaC_(n)H_(2n-3). An alkenyl group may comprise a terminal carbon-carbondouble bond and/or an internal carbon-carbon double bond. A terminalalkenyl group is an alkenyl group wherein a carbon-carbon double bond islocated at a terminal position of a carbon chain. An alkenyl group mayalso comprise two or more carbon-carbon double bonds. Examples of analkenyl group include ethenyl, propenyl, isopropenyl, t-butenyl,1,3-butadienyl, 1,3-pentadienyl, etc. Unless stated otherwise, analkenyl group may optionally be substituted with one or more,independently selected, substituents as defined below. Unless statedotherwise, an alkenyl group may optionally be interrupted by one or moreheteroatoms independently selected from the group consisting of O, N andS.

An alkynyl group comprises one or more carbon-carbon triple bonds, andmay be linear or branched. Unsubstituted alkynyl groups comprising oneC—C triple bond have the general formula C_(n)H_(2n-3). An alkynyl groupmay comprise a terminal carbon-carbon triple bond and/or an internalcarbon-carbon triple bond. A terminal alkynyl group is an alkynyl groupwherein a carbon-carbon triple bond is located at a terminal position ofa carbon chain. An alkynyl group may also comprise two or morecarbon-carbon triple bonds. Unless stated otherwise, an alkynyl groupmay optionally be substituted with one or more, independently selected,substituents as defined below. Examples of an alkynyl group includeethynyl, propynyl, isopropynyl, t-butynyl, etc. Unless stated otherwise,an alkynyl group may optionally be interrupted by one or moreheteroatoms independently selected from the group consisting of O, N andS.

An aryl group comprises six to twelve carbon atoms and may includemonocyclic and bicyclic structures. Optionally, the aryl group may besubstituted by one or more substituents further specified in thisdocument. Examples of aryl groups are phenyl and naphthyl.

Arylalkyl groups and alkylaryl groups comprise at least seven carbonatoms and may include monocyclic and bicyclic structures. Optionally,the arylalkyl groups and alkylaryl may be substituted by one or moresubstituents further specified in this document. An arylalkyl group isfor example benzyl. An alkylaryl group is for example 4-t-butylphenyl.

Heteroaryl groups comprise at least two carbon atoms (i.e. at least C₂)and one or more heteroatoms N, O, P or S. A heteroaryl group may have amonocyclic or a bicyclic structure. Optionally, the heteroaryl group maybe substituted by one or more substituents further specified in thisdocument. Examples of suitable heteroaryl groups include pyridinyl,quinolinyl, pyrimidinyl, pyrazinyl, pyrazolyl, imidazolyl, thiazolyl,pyrrolyl, furanyl, triazolyl, benzofuranyl, indolyl, purinyl,benzoxazolyl, thienyl, phospholyl and oxazolyl.

Heteroarylalkyl groups and alkylheteroaryl groups comprise at leastthree carbon atoms (i.e. at least C₃) and may include monocyclic andbicyclic structures. Optionally, the heteroaryl groups may besubstituted by one or more substituents further specified in thisdocument.

Where an aryl group is denoted as a (hetero)aryl group, the notation ismeant to include an aryl group and a heteroaryl group. Similarly, analkyl(hetero)aryl group is meant to include an alkylaryl group and aalkylheteroaryl group, and (hetero)arylalkyl is meant to include anarylalkyl group and a heteroarylalkyl group. A C₂-C₂₄ (hetero)aryl groupis thus to be interpreted as including a C₂-C₂₄ heteroaryl group and aC₆-C₂₄ aryl group. Similarly, a C₃-C₂₄ alkyl(hetero)aryl group is meantto include a C₇-C₂₄ alkylaryl group and a C₃-C₂₄ alkylheteroaryl group,and a C₃-C₂₄ (hetero)arylalkyl is meant to include a C₇-C₂₄ arylalkylgroup and a C₃-C₂₄ heteroarylalkyl group.

A cycloalkynyl group is a cyclic alkynyl group. An unsubstitutedcycloalkynyl group comprising one triple bond has the general formulaC_(n)H_(2n-5). Optionally, a cycloalkynyl group is substituted by one ormore substituents further specified in this document. An example of acycloalkynyl group is cyclooctynyl.

A heterocycloalkynyl group is a cycloalkynyl group interrupted byheteroatoms selected from the group of oxygen, nitrogen and sulphur.Optionally, a heterocycloalkynyl group is substituted by one or moresubstituents further specified in this document. An example of aheterocycloalkynyl group is azacyclooctynyl.

A (hetero)aryl group comprises an aryl group and a heteroaryl group. Analkyl(hetero)aryl group comprises an alkylaryl group and analkylheteroaryl group. A (hetero)arylalkyl group comprises a arylalkylgroup and a heteroarylalkyl groups. A (hetero)alkynyl group comprises analkynyl group and a heteroalkynyl group. A (hetero)cycloalkynyl groupcomprises an cycloalkynyl group and a heterocycloalkynyl group.

A (hetero)cycloalkyne compound is herein defined as a compoundcomprising a (hetero)cycloalkynyl group.

Several of the compounds disclosed in this description and in the claimsmay be described as fused (hetero)cycloalkyne compounds, i.e.(hetero)cycloalkyne compounds wherein a second ring structure is fused,i.e. annulated, to the (hetero)cycloalkynyl group. For example in afused (hetero)cyclooctyne compound, a cycloalkyl (e.g. a cyclopropyl) oran arene (e.g. benzene) may be annulated to the (hetero)cyclooctynylgroup. The triple bond of the (hetero)cyclooctynyl group in a fused(hetero)cyclooctyne compound may be located on either one of the threepossible locations, i.e. on the 2, 3 or 4 position of the cyclooctynemoiety (numbering according to “IUPAC Nomenclature of OrganicChemistry”, Rule A31.2). The description of any fused(hetero)cyclooctyne compound in this description and in the claims ismeant to include all three individual regioisomers of the cyclooctynemoiety.

Unless stated otherwise, alkyl groups, cycloalkyl groups, alkenylgroups, alkynyl groups, (hetero)aryl groups, (hetero)arylalkyl groups,alkyl(hetero)aryl groups, alkylene groups, alkenylene groups, alkynylenegroups, cycloalkylene groups, cycloalkenylene groups, cycloalkynylenegroups, (hetero)arylene groups, alkyl(hetero)arylene groups,(hetero)arylalkylene groups, (hetero)arylalkenylene groups,(hetero)arylalkynylene groups, alkenyl groups, alkoxy groups, alkenyloxygroups, (hetero)aryloxy groups, alkynyloxy groups and cycloalkyloxygroups may be substituted with one or more substituents independentlyselected from the group consisting of C₁-C₁₂ alkyl groups, C₂-C₁₂alkenyl groups, C₂-C₁₂ alkynyl groups, C₃-C₁₂ cycloalkyl groups, C₅-C₁₂cycloalkenyl groups, C₆-C₁₂ cycloalkynyl groups, C₁-C₁₂ alkoxy groups,C₂-C₁₂ alkenyloxy groups, C₂-C₁₂ alkynyloxy groups, C₃-C₁₂ cycloalkyloxygroups, halogens, amino groups, oxo and silyl groups, wherein the silylgroups can be represented by the formula (R²⁰)₃Si—, wherein R²⁰ isindependently selected from the group consisting of C₁-C₁₂ alkyl groups,C₂-C₁₂ alkenyl groups, C₂-C₁₂ alkynyl groups, C₃-C₁₂ cycloalkyl groups,C₁-C₁₂ alkoxy groups, C₂-C₁₂ alkenyloxy groups, C₂-C₁₂ alkynyloxy groupsand C₃-C₁₂ cycloalkyloxy groups, wherein the alkyl groups, alkenylgroups, alkynyl groups, cycloalkyl groups, alkoxy groups, alkenyloxygroups, alkynyloxy groups and cycloalkyloxy groups are optionallysubstituted, the alkyl groups, the alkoxy groups, the cycloalkyl groupsand the cycloalkoxy groups being optionally interrupted by one of morehetero-atoms selected from the group consisting of O, N and S.

The general term “sugar” is herein used to indicate a monosaccharide,for example glucose (Glc), galactose (Gal), mannose (Man) and fucose(Fuc). The term “sugar derivative” is herein used to indicate aderivative of a monosaccharide sugar, i.e. a monosaccharide sugarcomprising substituents and/or functional groups. Examples of a sugarderivative include amino sugars and sugar acids, e.g. glucosamine(GlcNH₂), galactosamine (GalNH₂)N-acetylglucosamine (GlcNAc),N-acetylgalactosamine (GalNAc), sialic acid (Sia) which is also referredto as N-acetylneuraminic acid (NeuNAc), and N-acetylmuramic acid(MurNAc), glucuronic acid (GlcA) and iduronic acid (IdoA).

The term “nucleotide” is herein used in its normal scientific meaning.The term “nucleotide” refers to a molecule that is composed of anucleobase, a five-carbon sugar (either ribose or 2-deoxyribose), andone, two or three phosphate groups. Without the phosphate group, thenucleobase and sugar compose a nucleoside. A nucleotide can thus also becalled a nucleoside monophosphate, a nucleoside diphosphate or anucleoside triphosphate. The nucleobase may be adenine, guanine,cytosine, uracil or thymine. Examples of a nucleotide include uridinediphosphate (UDP), guanosine diphosphate (GDP), thymidine diphosphate(TDP), cytidine diphosphate (CDP) and cytidine monophosphate (CMP).

The term “protein” is herein used in its normal scientific meaning.Herein, polypeptides comprising about 10 or more amino acids areconsidered proteins. A protein may comprise natural, but also unnaturalamino acids.

The term “glycoprotein” is herein used in its normal scientific meaningand refers to a protein comprising one or more monosaccharide oroligosaccharide chains (“glycans”) covalently bonded to the protein. Aglycan may be attached to a hydroxyl group on the protein(O-linked-glycan), e.g. to the hydroxyl group of serine, threonine,tyrosine, hydroxylysine or hydroxyproline, or to an amide function onthe protein (N-glycoprotein), e.g. asparagine or arginine, or to acarbon on the protein (C-glycoprotein), e.g. tryptophan. A glycoproteinmay comprise more than one glycan, may comprise a combination of one ormore monosaccharide and one or more oligosaccharide glycans, and maycomprise a combination of N-linked, O-linked and C-linked glycans. It isestimated that more than 50% of all proteins have some form ofglycosylation and therefore qualify as glycoprotein. Examples ofglycoproteins include PSMA (prostate-specific membrane antigen), CAL(candida antartica lipase), gp41, gp120, EPO (erythropoietin),antifreeze protein and antibodies.

The term “glycan” is herein used in its normal scientific meaning andrefers to a monosaccharide or oligosaccharide chain that is linked to aprotein. The term glycan thus refers to the carbohydrate-part of aglycoprotein. The glycan is attached to a protein via the C-1 carbon ofone sugar, which may be without further substitution (monosaccharide) ormay be further substituted at one or more of its hydroxyl groups(oligosaccharide). A naturally occurring glycan typically comprises 1 toabout 10 saccharide moieties. However, when a longer saccharide chain islinked to a protein, said saccharide chain is herein also considered aglycan. A glycan of a glycoprotein may be a monosaccharide. Typically, amonosaccharide glycan of a glycoprotein consists of a singleN-acetylglucosamine (GlcNAc), glucose (Glc), mannose (Man) or fucose(Fuc) covalently attached to the protein. A glycan may also be anoligosaccharide. An oligosaccharide chain of a glycoprotein may belinear or branched. In an oligosaccharide, the sugar that is directlyattached to the protein is called the core sugar. In an oligosaccharide,a sugar that is not directly attached to the protein and is attached toat least two other sugars is called an internal sugar. In anoligosaccharide, a sugar that is not directly attached to the proteinbut to a single other sugar, i.e. carrying no further sugar substituentsat one or more of its other hydroxyl groups, is called the terminalsugar. For the avoidance of doubt, there may exist multiple terminalsugars in an oligosaccharide of a glycoprotein, but only one core sugar.A glycan may be an O-linked glycan, an N-linked glycan or a C-linkedglycan. In an O-linked glycan a monosaccharide or oligosaccharide glycanis bonded to an O-atom in an amino acid of the protein, typically via ahydroxyl group of serine (Ser) or threonine (Thr). In an N-linked glycana monosaccharide or oligosaccharide glycan is bonded to the protein viaan N-atom in an amino acid of the protein, typically via an amidenitrogen in the side chain of asparagine (Asn) or arginine (Arg). In aC-linked glycan a monosaccharide or oligosaccharide glycan is bonded toa C-atom in an amino acid of the protein, typically to a C-atom oftryptophan (Trp).

The term “antibody” is herein used in its normal scientific meaning. Anantibody is a protein generated by the immune system that is capable ofrecognizing and binding to a specific antigen. An antibody is an exampleof a glycoprotein. The term antibody herein is used in its broadestsense and specifically includes monoclonal antibodies, polyclonalantibodies, dimers, multimers, multi-specific antibodies (e.g.bispecific antibodies), antibody fragments, and double and single chainantibodies. The term “antibody” is herein also meant to include humanantibodies, humanized antibodies, chimeric antibodies and antibodiesspecifically binding cancer antigen. The term “antibody” is meant toinclude whole antibodies, but also antigen-binding fragments of anantibody, for example an antibody Fab fragment, F(ab′)₂, Fv fragment orFc fragment from a cleaved antibody, a scFv-Fc fragment, a minibody, adiabody or a scFv. Furthermore, the term includes genetically engineeredantibodies and derivatives of an antibody. Antibodies, fragments ofantibodies and genetically engineered antibodies may be obtained bymethods that are known in the art. Typical examples of antibodiesinclude, amongst others, abciximab, rituximab, basiliximab, palivizumab,infliximab, trastuzumab, alemtuzumab, adalimumab, tositumomab-1131,cetuximab, ibrituximab tiuxetan, omalizumab, bevacizumab, natalizumab,ranibizumab, panitumumab, eculizumab, certolizumab pegol, golimumab,canakinumab, catumaxomab, ustekinumab, tocilizumab, ofatumumab,denosumab, belimumab, ipilimumab and brentuximab.

A linker is herein defined as a moiety that connects two or moreelements of a compound. For example in a bioconjugate, a biomolecule anda target molecule are covalently connected to each other via a linker;in the linker-conjugate a reactive group Q¹ is covalently connected to atarget molecule via a linker; in a linker-construct a reactive group Q¹is covalently connected to a reactive group Q² via a linker. A linkermay comprise one or more spacer-moieties.

A spacer-moiety is herein defined as a moiety that spaces (i.e. providesdistance between) and covalently links together two (or more) parts of alinker. The linker may be part of e.g. a linker-construct, thelinker-conjugate or a bioconjugate, as defined below.

A linker-construct is herein defined as a compound wherein a reactivegroup Q¹ is covalently connected to a reactive group Q² via a linker. Alinker-construct comprises a reactive group Q¹ capable of reacting witha reactive group present on a biomolecule, and a reactive group Q²capable of reacting with a reactive group present on a target molecule.Q¹ and Q² may be the same, or different. A linker-construct may alsocomprise more than one reactive group Q¹ and/or more than one reactivegroup Q². A linker-construct may also be denoted as Q¹-Sp-Q², wherein Q¹is a reactive group capable of reacting with a reactive group F¹ presenton a biomolecule, Q² is a reactive group capable of reacting with areactive group F² present on a target molecule and Sp is a spacermoiety. When a linker-construct comprises more than one reactive groupQ¹ and/or more than one reactive group Q², the linker-construct may bedenoted as (Q¹)_(y)-Sp-(Q²)_(z), wherein y is an integer in the range of1 to 10 and z is an integer in the range of 1 to 10. Preferably, y is 1,2, 3 or 4, more preferably y is 1 or 2 and most preferably, y is 1.Preferably, z is 1, 2, 3, 4, 5 or 6, more preferably z is 1, 2, 3 or 4,even more preferably z is 1, 2 or 3, yet even more preferably z is 1 or2 and most preferably z is 1. More preferably, y is 1 or 2, preferably1, and z is 1, 2, 3 or 4, yet even more preferably y is 1 or 2,preferably 1, and z is 1, 2 or 3, yet even more preferably y is 1 or 2,preferably 1, and z is 1 or 2, and most preferably y is 1 and z is 1.

A bioconjugate is herein defined as a compound wherein a biomolecule iscovalently connected to a target molecule via a linker. A bioconjugatecomprises one or more biomolecules and/or one or more target molecules.The linker may comprise one or more spacer moieties.

When a compound is herein referred to as a compound comprising analpha-end and an omega-end, said compound comprises two (or more) ends,the first end being referred to as the alpha-end and the second endbeing referred to as the omega-end. Said compound may comprise more thantwo ends, i.e. a third, fourth etc. end may be present in the compound.

A biomolecule is herein defined as any molecule that can be isolatedfrom nature or any molecule composed of smaller molecular buildingblocks that are the constituents of macromolecular structures derivedfrom nature, in particular nucleic acids, proteins, glycans and lipids.Examples of a biomolecule include an enzyme, a (non-catalytic) protein,a polypeptide, a peptide, an amino acid, an oligonucleotide, amonosaccharide, an oligosaccharide, a polysaccharide, a glycan, a lipidand a hormone.

A target molecule, also referred to as a molecule of interest (MOI), isherein defined as molecular structure possessing a desired property thatis imparted onto the biomolecule upon conjugation. Herein, a sulfamidelinker and conjugates of said sulfamide linker are disclosed. The term“sulfamide linker” refers to a linker comprising a sulfamide group, moreparticularly an acylsulfamide group [—C(O)—N(H)—S(O)₂—N(R¹)—] and/or acarbamoyl sulfamide group [—O—C(O)—N(H)—S(O)₂—N(R¹)—].

Herein, the term “therapeutic index” (TI) has the conventional meaningwell known to a person skilled in the art, and refers to the ratio ofthe dose of drug that is toxic (i.e. causes adverse effects at anincidence or severity not compatible with the targeted indication) for50% of the population (TD₅₀) divided by the dose that leads to thedesired pharmacological effect in 50% of the population (effective doseor ED₅₀). Hence, TI=TD₅₀/ED₅₀. The therapeutic index may be determinedby clinical trials or for example by plasma exposure tests. See alsoMuller, et al. Nature Reviews Drug Discovery 2012, 11, 751-761.

Herein, the term “therapeutic efficacy” denotes the capacity of asubstance to achieve a certain therapeutic effect, e.g. reduction intumour volume. Therapeutic effects can be measured determining theextent in which a substance can achieve the desired effect, typically incomparison with another substance under the same circumstances. Asuitable measure for the therapeutic efficacy is the ED₅₀ value, whichmay for example be determined during clinical trials or by plasmaexposure tests. In case of preclinical therapeutic efficacydetermination, the therapeutic effect of a bioconjugate (e.g. an ADC),can be validated by patient-derived tumour xenografts in mice in whichcase the efficacy refers to the ability of the ADC to provide abeneficial effect. Alternatively the tolerability of said ADC in arodent safety study can also be a measure of the therapeutic effect.

Herein, the term “tolerability” refers to the maximum dose of a specificsubstance that does not cause adverse effects at an incidence orseverity not compatible with the targeted indication. A suitable measurefor the tolerability for a specific substance is the TD₅₀ value, whichmay for example be determined during clinical trials or by plasmaexposure tests.

Linker

A linker is herein defined as a moiety that connects two or moreelements of a compound. For example in a bioconjugate, a biomolecule anda target molecule are covalently connected to each other via a linker;in a linker-conjugate a reactive group Q¹ is covalently connected to atarget molecule via a linker; in a linker-construct a reactive group Q¹is covalently connected to a reactive group Q² via a linker.

In the context of the present invention, the linker-construct,linker-conjugate and bioconjugate according to the invention comprise abranched sulfamide linker. The term “branched sulfamide linker” refersto a linker comprising a sulfamide group, more particularly anacylsulfamide group [—C(O)—N(H)—S(O)₂—N(R¹)—] and/or a carbamoylsulfamide group [—O—C(O)—N(H)—S(O)₂—N(R¹)—]. The linker according to theinvention further comprises branching moiety (BM).

Branching Moiety BM

A “branching moiety” in the context of the present invention refers to amoiety that is embedded in a linker connecting two moieties, inparticular a target molecule D and a reactive group Q¹, or a targetmolecule D and a biomolecule B, or a reactive group Q¹ and a reactivegroup Q², and further comprises a branch points that connects to thesulfamide group according to the present invention. In other words, thebranching moiety comprises at least three bonds to other moieties, onebond to reactive group Q² or target molecule D, optionally via a spacer,one bond to reactive group Q¹ or biomolecule B, optionally via a spacer,and one bond to sulfamide group SG, optionally via a spacer. Branchingmoiety BM may contain additional bonds to other moieties, such as two ormore bonds to target molecules, two or more bonds to reactive groups orbiomolecules and/or two or more bonds to sulfamide groups. In oneembodiment, the branching moiety comprises 3, 4, 5 or 6 bonds to othermoieties, preferably 3 or 4 bonds to other moieties. In case thebranching moiety contains more than 3 bonds to other moieties, it ispreferred that two or more bonds are to a target molecule.

Any moiety that contains at least three bonds to other moieties issuitable as branching moiety in the context of the present invention.Suitable branching moieties include a carbon atom (BM-1 and BM-2), anitrogen atom (amine (BM-3) and ammonium (BM-4)), a phosphorus atom(phosphine (BM-5) and phosphine oxide (BM-6)), aromatic rings such as aphenyl ring (e.g. BM-7 and BM-8) or a pyridyl ring (e.g. BM-9 andBM-10), a (hetero)cycle (e.g. BM-11 and BM-12) and polycyclic moieties(e.g. BM-13, BM-14 and BM-15). Preferred branching moieties are selectedfrom carbon atoms and phenyl rings, most preferably BM is a carbon atom.Structures (BM-1) to (BM-15) are depicted here below, wherein the threeor four branches, i.e. bonds to other moieties as defined above, areindicated by

(a bond labelled with *).

In (BM-1), one of the branches labelled with * may be a single or adouble bond, indicated with

. In (BM-4), An⁻ is an anion, typically a pharmaceutically acceptableanion. In (BM-11) to (BM-15), the following applies:

-   -   each of p, q and q is individually an integer in the range of        0-5, preferably 0 or 1, most preferably 1;    -   each of W¹, W² and W³ is independently selected from C(R²¹)_(r)        and N;    -   each of W⁴, W⁵ and W⁶ is independently selected from        C(R²¹)_(r+1), N(R²²)_(r), O and S;    -   each        represents a single or double bond;    -   r is 0 or 1 or 2, preferably 0 or 1;    -   each R²¹ is independently selected from the group consisting of        hydrogen, OH, C₁-C₂₄ alkyl groups, C₁-C₂₄ alkoxy groups, C₃-C₂₄        cycloalkyl groups, C₂-C₂₄ (hetero)aryl groups, C₃-C₂₄        alkyl(hetero)aryl groups and C₃-C₂₄ (hetero)arylalkyl groups,        wherein the C₁-C₂₄ alkyl groups, C₁-C₂₄ alkoxy groups, C₃-C₂₄        cycloalkyl groups, C₂-C₂₄ (hetero)aryl groups, C₃-C₂₄        alkyl(hetero)aryl groups and C₃-C₂₄ (hetero)arylalkyl groups are        optionally substituted and optionally interrupted by one or more        heteroatoms selected from O, S and NR³ wherein R³ is        independently selected from the group consisting of hydrogen and        C₁-C₄ alkyl groups, or R²¹ is a further branch        , connected to a further occurrence of SG, D, Q¹, Q² or B,        optionally via a spacer; and    -   each R²² is independently selected from the group consisting of        hydrogen, C₁-C₂₄ alkyl groups, C₃-C₂₄ cycloalkyl groups, C₂-C₂₄        (hetero)aryl groups, C₃-C₂₄ alkyl(hetero)aryl groups and C₃-C₂₄        (hetero)arylalkyl groups, wherein the C₁-C₂₄ alkyl groups,        C₁-C₂₄ alkoxy groups, C₃-C₂₄ cycloalkyl groups, C₂-C₂₄        (hetero)aryl groups, C₃-C₂₄ alkyl(hetero)aryl groups and C₃-C₂₄        (hetero)arylalkyl groups are optionally substituted and        optionally interrupted by one or more heteroatoms selected from        O, S and NR³ wherein R³ is independently selected from the group        consisting of hydrogen and C₁-C₄ alkyl groups, or R²² is a        further branch        , connected to a further occurrence of SG, D, Q¹, Q² or B,        optionally via a spacer

The skilled person appreciates that the values of r and the bond orderof the bonds represented by

are interdependent. Thus, whenever an occurrence of W is bonded to anendocyclic double bond, r=1 for that occurrence of W, while whenever anoccurrence of W is bonded to two endocyclic single bonds, r=0 for thatoccurrence of W. For BM-12, at least one of o and p is not 0.

Representative examples of branching moieties according to structure(BM-11) and (BM-12) include cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, cyclooctyl, cyclopropenyl, cyclobutenyl,cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, aziridine,azetidine, diazetidine, oxetane, thietane, pyrrolidine, dihydropyrrolyl,tetrahydrofuranyl, dihydrofuranyl, thiolanyl, imidazolinyl,pyrazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl,isothiazolidinyl, dioxolanyl, dithiolanyl, piperidinyl, oxanyl, thianyl,piperazinyl, morpholino, thiomorpholino, dioxanyl, trioxanyl, dithyanyl,trithianyl, azepanyl, oxepanyl and thiepanyl. Preferred cyclic moietiesfor use as branching moiety include cyclopropenyl, cyclohexyl, oxanyland dioxanyl. The substitution pattern of the three branches determineswhether the branching moiety is of structure (BM-11) or of structure(BM-12). In one embodiment, BM is not cyclopropyl. In one embodiment, BMis not pyrrolidine.

Representative examples of branching moieties according to structure(BM-13) to (BM-15) include decalin, tetralin, dialin, naphthalene,indene, indane, isoindene, indole, isoindole, indoline, isoindoline, andthe like.

At least one, preferably one or two, of the branches indicated with * instructures (BM-1) to (BM-15) is connected to reactive group Q² or targetmolecule D. At least one, preferably one, of the branches indicatedwith * in structures (BM-1) to (BM-15) is connected to reactive group Q¹or biomolecule B. At least one, preferably one or two, of the branchesindicated with * in structures (BM-1) to (BM-15) is connected tosulfamide group SG.

In case a branching moiety contains four branches (bonds labelled with*), two of those may be connected or may be part of a single occurrenceof SG, D, Q¹, Q² or B, via a cyclic moiety. For example, in case thebranching moiety is the carbon atom of BM-2, one bond may be to SG, onebond may be to D and two bonds may form a cycle, e.g. a cyclopropylring, that is part of Q¹, for example part of a cyclooctyne ring.

In a preferred embodiment, BM is a carbon atom. In case the carbon atomis according to structure (BM-2) and two of the branches indicatedwith * are connected to identical moieties, than the carbon atom is notchiral. Such two identical moieties may be two target molecules D,optionally linked via a spacer, two biomolecules B, optionally linkedvia a spacer, or two sulfamide groups SG-E, optionally linked via aspacer, preferably two target molecules D, optionally linked via aspacer. However, in case the carbon atom is according to structure(BM-1) or is according to structure (BM-2) and all four bonds are todistinct moieties, e.g. when two target molecules D, optionally linkedvia a spacer, are linked to the branching moiety according to structure(BM-2), wherein the target molecules and/or the spacer differ, thecarbon atom is chiral. The stereochemistry of the carbon atom is notcrucial for the present invention, and may be S or R. The same holds forthe quaternary ammonium ion of (BM-4) and phosphine (BM-6). Mostpreferably, the carbon atom is according to structure (BM-1).

One of the branches indicated with * in the carbon atom according tostructure (BM-1) may be a double bond, in which case the carbon atom maybe part of an alkene or imine. In case BM is a carbon atom, the carbonatom may be part of a larger functional group, such as an acetal, aketal, a hemiketal, an orthoester, an orthocarbonate ester, and thelike. This also holds in case BM is a nitrogen or phosphorus atom, inwhich case it may be part of an amide, an imide, an imine, a phosphineoxide (as in BM-6) or a phosphotriester.

In a preferred embodiment, BM is a phenyl ring. Phenyl rings may betri-, tetra-, penta- or hexa-substituted, preferably they are tri- ortetra-substituted, i.e. according to structure (BM-7) or structure(BM-8), respectively. Most preferably, the phenyl ring is according tostructure (BM-7).

The substitution pattern of the phenyl ring may be of anyregiochemistry, such as 1,2,3-substituted phenyl rings,1,2,4-substituted phenyl rings, 1,3,5-substituted phenyl rings,1,2,3,4-substituted phenyl rings, 1,2,3,5-substituted phenyl rings or1,2,4,5-substituted phenyl rings. To allow optimal flexibility andconformational freedom, it is preferred that the phenyl ring isaccording to structure (BM-7), most preferably the phenyl ring is1,3,5-substituted. The same holds for the pyridine rings of (BM-9) and(BM-10).

Sulfamide Group SG

In the linker of the present invention, branching moiety BM is connectedto the sulfamide group SG according to formula (1). Herein, the bonds tothe other moieties of the compounds according to the inventions areindicated by 1 and 2. Sulfamide group SG is connected via one of thebonds indicated with 1 and 2 to branching moiety BM, optionally via aspacer, and with the other bond indicated with 1 or 2 to capping groupE, optionally via a spacer.

Herein:

-   -   a is 0 or 1; and    -   R¹ is selected from the group consisting of hydrogen, C₁-C₂₄        alkyl groups, C₃-C₂₄ cycloalkyl groups, C₂-C₂₄ (hetero)aryl        groups, C₃-C₂₄ alkyl(hetero)aryl groups and C₃-C₂₄        (hetero)arylalkyl groups, wherein the C₁-C₂₄ alkyl groups,        C₃-C₂₄ cycloalkyl groups, C₂-C₂₄ (hetero)aryl groups, C₃-C₂₄        alkyl(hetero)aryl groups and C₃-C₂₄ (hetero)arylalkyl groups are        optionally substituted and optionally interrupted by one or more        heteroatoms selected from O, S and NR³ wherein R³ is        independently selected from the group consisting of hydrogen and        C₁-C₄ alkyl groups, or R¹ is a further target molecule D,        wherein the target molecule is optionally connected to N via a        spacer moiety.

The linker according to the invention comprises a group according toformula (1) as defined above. In a preferred embodiment, the linkeraccording to the invention comprises a group according to formula (1)wherein a is 0, or a salt thereof. In this embodiment, the compound thuscomprises a group according to formula (2) or a salt thereof:

wherein R¹ is as defined above.

In another preferred embodiment, the linker according to the inventioncomprises a group according to formula (1) wherein a is 1. In thisembodiment, the linker thus comprises a group according to formula (3)or a salt thereof:

wherein R¹ is as defined above.

In the groups according to formula (1), (2) and (3), R¹ is selected fromthe group consisting of hydrogen, C₁-C₂₄ alkyl groups, C₃-C₂₄ cycloalkylgroups, C₂-C₂₄ (hetero)aryl groups, C₃-C₂₄ alkyl(hetero)aryl groups andC₃-C₂₄ (hetero)arylalkyl groups, the C₁-C₂₄ alkyl groups, C₃-C₂₄cycloalkyl groups, C₂-C₂₄ (hetero)aryl groups, C₃-C₂₄ alkyl(hetero)arylgroups and C₃-C₂₄ (hetero)arylalkyl groups optionally substituted andoptionally interrupted by one or more heteroatoms selected from O, S andNR³ wherein R³ is independently selected from the group consisting ofhydrogen and C₁-C₄ alkyl groups, or R¹ is a target molecule D, whereinthe target molecule is optionally connected to N via a spacer moiety. Inone embodiment, in case R¹ is a target molecule D, optionally connectedvia a spacer, D is not a drug or a prodrug, preferably not an activesubstance. In one embodiment, R¹ is not D.

In a preferred embodiment, R¹ is hydrogen or a C₁-C₂₀ alkyl group, morepreferably R¹ is hydrogen or a C₁-C₁₆ alkyl group, even more preferablyR¹ is hydrogen or a C₁-C₁₀ alkyl group, wherein the alkyl group isoptionally substituted and optionally interrupted by one or moreheteroatoms selected from O, S and NR³, preferably O, wherein R³ isindependently selected from the group consisting of hydrogen and C₁-C₄alkyl groups. In a preferred embodiment, R¹ is hydrogen. In anotherpreferred embodiment, R¹ is a C₁-C₂₀ alkyl group, more preferably aC₁-C₁₆ alkyl group, even more preferably a C₁-C₁₀ alkyl group, whereinthe alkyl group is optionally interrupted by one or more O-atoms, andwherein the alkyl group is optionally substituted with an —OH group,preferably a terminal —OH group. In this embodiment, it is furtherpreferred that R¹ is a (poly)ethylene glycol chain comprising a terminal—OH group. In another preferred embodiment, R¹ is selected from thegroup consisting of hydrogen, methyl, ethyl, n-propyl, i-propyl,n-butyl, s-butyl and t-butyl, more preferably from the group consistingof hydrogen, methyl, ethyl, n-propyl and i-propyl, and even morepreferably from the group consisting of hydrogen, methyl and ethyl. Yeteven more preferably R¹ is hydrogen or methyl, and most preferably R¹ ishydrogen.

In another preferred embodiment, R¹ is a target molecule D. Optionally,the target molecule D is connected to N via one or more spacer-moieties.The spacer-moiety, if present, is defined as a moiety that spaces, i.e.provides a certain distance between, and covalently links targetmolecule D and N. The target molecule D and preferred embodimentsthereof are described in more detail below.

Capping Group E

Sulfamide group SG is connected to capping group E, optionally via aspacer moiety. Any group or moiety known in the art to act as cappinggroup may be used as capping group E. Suitable capping groups includehydrogen, C₁-C₂₄ alkyl groups, C₃-C₂₄ cycloalkyl groups, C₂-C₂₄(hetero)aryl groups, C₃-C₂₄ alkyl(hetero)aryl groups, C₃-C₂₄(hetero)arylalkyl groups, polyethylene glycol groups represented by—(CH₂CH₂O)_(p)CH₃ (wherein p=1-10, preferably 2-4), wherein the C₁-C₂₄alkyl groups, C₃-C₂₄ cycloalkyl groups, C₂-C₂₄ (hetero)aryl groups,C₃-C₂₄ alkyl(hetero)aryl groups and C₃-C₂₄ (hetero)arylalkyl groups areoptionally substituted and optionally interrupted by one or moreheteroatoms selected from O, S and NR³ wherein R³ is independentlyselected from the group consisting of hydrogen and C₁-C₄ alkyl groups,or E is a further target molecule D. In one embodiment, in case E is atarget molecule D, D is not a drug or a prodrug, preferably not anactive substance. In one embodiment, E is not D.

In one embodiment, both R¹ and E are further target molecules D, whichmay be the same or different and the same or different as the targetmolecule D in the main chain. In one embodiment, one of R¹ and E is afurther target molecule D, which may be the same or different as thetarget molecule D in the main chain. In one embodiment, both R¹ and Eare not a further target molecule D.

In one embodiment, E is a polyethylene glycol groups, which is typicallyrepresented by —(CH₂CH₂O)_(s)CH₃, wherein s is an integer in the rangeof 1-10, preferably 2-4. In one embodiment, E is a C₁-C₂₄ alkyl group,C₂-C₂₄ (hetero)aryl group or a C₃-C₂₄ alkyl(hetero)aryl group,preferably a C₆-C₁₂ alkaryl group, most preferably a benzyl group.

The linker according to the present invention connects reactive group Q²or target molecule D on one hand, and reactive group Q¹ or biomolecule Bon the other hand.

Preferred Linkers

Sulfamide linkers according to the present invention are preferablyrepresented by formula (2):

Herein:

-   -   BM is a branching moiety as defined above;    -   E is a capping group as defined above;    -   SG is a sulfamide group according to formula (1), wherein a and        R¹ are as defined above;    -   b is independently 0 or 1;    -   c is 0 or 1;    -   d is 0 or 1;    -   e is 0 or 1;    -   f is an integer in the range of 1 to 10;    -   g is 0 or 1;    -   i is 0 or 1;    -   k is 0 or 1;    -   l is 0 or 1;    -   Sp¹ is a spacer moiety;    -   Sp² is a spacer moiety;    -   Sp³ is a spacer moiety;    -   Sp⁴ is a spacer moiety;    -   Sp⁵ is a spacer moiety;    -   Sp⁶ is a spacer moiety;    -   Z¹ is a connecting group; and    -   Z² is a connecting group.

The bonds labelled with * in structure (2) are the bonds to the othermoieties that are connected by the linker within the compounds accordingto the invention.

As will be understood by the person skilled in the art, preferredembodiments of the linker of structure (2) will depend on e.g. thenature of reactive groups Q¹ and D in the linker-conjugate, thesynthetic method to prepare the linker-construct and linker-conjugate(e.g. the nature of complementary functional group F² on a targetmolecule), the nature of a bioconjugate that is prepared using thelinker-conjugate (e.g. the nature of complementary functional group F¹on the biomolecule).

When Q¹ is for example a cyclooctynyl group according to formula (9n),(9o), (9p), (9q) or (9zk) as defined below, then preferably Sp³ ispresent (g is 1). When for example the linker-conjugate was prepared viareaction of a reactive group Q² that is a cyclooctynyl group accordingto formula (9n), (9o), (9p), (9q) or (9zk) with an azido functionalgroup F², then preferably Sp⁴ is present (i is 1). Furthermore, it ispreferred that at least one of Sp¹, Sp², Sp³ and Sp⁴ is present, i.e. atleast one of b, c, g, and i is not 0. In another preferred embodiment,at least one of Sp¹ and Sp⁴ and at least one of Sp² and Sp³ are present.When f is 2 or more, it is preferred that Sp¹ is present (b is 1).Preferred embodiments of Sp¹, Sp², Sp³ and Sp⁴ are as defined below.

As defined above, Z¹ is a connecting group that connects Q¹ or B or Sp³to Sp² or BM, and Z² is a connecting group that connects Q² or D or Sp⁴to Sp¹ or BM. As described in more detail above, the term “connectinggroup” refers to a structural element connecting one part of a compoundand another part of the same compound.

In a compound according to formula (2), connecting group Z¹, whenpresent (i.e. when d is 1), connects Q¹ or B (optionally via a spacermoiety Sp³) to branching moiety BM, optionally via a spacer moiety Sp².More particularly, when Z¹ is present (i.e. d is 1), and when Sp³ andSp² are absent (i.e. g is 0 and c is 0), Z¹ connects Q¹ or B to BM ofthe linker according to formula (2). When Z¹ is present (i.e. when d is1), Sp³ is present (i.e. g is 1) and Sp² is absent (i.e. c is 0), Z¹connects spacer moiety Sp³ to BM of the linker according to formula (2).When Z¹ is present (i.e. d is 1), and when Sp³ and Sp² are present (i.e.g is 1 and c is 1), Z¹ connects spacer moiety Sp³ to spacer moiety Sp²of the linker according to formula (2). When Z¹ is present (i.e. when dis 1), Sp³ is absent (i.e. g is 0) and Sp² is present (i.e. c is 1), Z¹connects Q¹ or B to spacer moiety Sp² of the linker according to formula(2). In the compound according to formula (2), when c, d and g are all0, then Q¹ or B is attached directly to BM of the linker according toformula (2).

In a compound according to formula (2), connecting group Z², whenpresent (i.e. when e is 1), connects D or Q² (optionally via a spacermoiety Sp⁴) to BM of the linker according to formula (2), optionally viaa spacer moiety Sp¹. More particularly, when Z² is present (i.e. e is1), and when Sp¹ and Sp⁴ are absent (i.e. b is 0 and i is 0), Z²connects D or Q² to BM of the linker according to formula (2). When Z²is present (i.e. when e is 1), Sp⁴ is present (i.e. i is 1) and Sp¹ isabsent (i.e. b is 0), Z² connects spacer moiety Sp⁴ to BM of the linkeraccording to formula (2). When Z² is present (i.e. e is 1), and when Sp⁴and Sp¹ are present (i.e. b is 1 and i is 1), Z² connects spacer moietySp⁴ to spacer moiety Sp¹ of the linker according to formula (2). When Z²is present (i.e. when e is 1), Sp⁴ is absent (i.e. i is 0) and Sp¹ ispresent (i.e. c is b), Z² connects D or Q² to spacer moiety Sp¹ of thelinker according to formula (2). In the compound according to formula(2), when b, e and i are all 0, then D or Q² is attached directly to BMof the linker according to formula (2).

As will be understood by the person skilled in the art, the nature of aconnecting group depends on the type of organic reaction with which theconnection between the specific parts of said compound was obtained. Alarge number of organic reactions are available for connecting areactive group Q¹ to a spacer moiety, and for connecting a targetmolecule to a spacer-moiety. Consequently, there is a large variety ofconnecting groups Z¹ and Z².

In a preferred embodiment of the linker according to formula (2), Z¹ andZ² are independently selected from the group consisting of —O—, —S—,—S—S—, —NR²—, —N═N—, —C(O)—, —C(O)—NR²—, —O—C(O)—, —O—C(O)—O—,—O—C(O)—NR², —NR²—C(O)—, —NR²—C(O)—O—, —NR²—C(O)—NR²—, —S—C(O)—,—S—C(O)—O—, —S—C(O)—NR²—, —S(O)—, —S(O)₂—, —O—S(O)₂—, —O—S(O)₂—O—,—O—S(O)₂—NR²—, —O—S(O)—, —O—S(O)—O—, —O—S(O)—NR²—, —O—NR²—C(O)—,—O—NR²—C(O)—O—, —O—NR²—C(O)—NR²—, —NR²—O—C(O)—, —NR²—O—C(O)—O—,—NR²—O—C(O)—NR²—, —O—NR²—C(S)—, —O—NR²—C(S)—O—, —O—NR²—C(S)—NR²—,—NR²—O—C(S)—, —NR²—O—C(S)—O—, —NR²—O—C(S)—NR²—, —O—C(S)—, —O—C(S)—O—,—O—C(S)—NR²—, —NR²—C(S)—, —NR²—C(S)—O—, —NR²—C(S)—NR²—, —S—S(O)₂—,—S—S(O)₂—O—, —S—S(O)₂—NR²—, —NR²—O—S(O)—, —NR²—O—S(O)—O—,—NR²—O—S(O)—NR²—, —NR²—O—S(O)₂—, —NR²—O—S(O)₂—O—, —NR²—O—S(O)₂—NR²—,—O—NR²—S(O)—, —O—NR²—S(O)—O—, —O—NR²—S(O)—NR²—, —O—NR²—S(O)₂—O—,—O—NR²—S(O)₂—NR²—, —O—NR²—S(O)₂—, —O—P(O)(R²)₂—, —S—P(O)(R²)₂—,—NR²—P(O)(R²)₂— and combinations of two or more thereof, wherein R² isindependently selected from the group consisting of hydrogen, C₁-C₂₄alkyl groups, C₂-C₂₄ alkenyl groups, C₂-C₂₄ alkynyl groups and C₃-C₂₄cycloalkyl groups, the alkyl groups, alkenyl groups, alkynyl groups andcycloalkyl groups being optionally substituted.

As described above, in the compound according to formula (2), Sp¹, Sp²,Sp³, Sp⁴, Sp⁵ and Sp⁶ are spacer-moieties. Sp¹, Sp², Sp³, Sp⁴, Sp⁵ andSp⁶ may be, independently, absent or present (b, c, g, i, k and l are,independently, 0 or 1). Each spacer, if present, may be different fromany other spacer, if present.

Spacer-moieties are known to a person skilled in the art. Examples ofsuitable spacer-moieties include (poly)ethylene glycol diamines (e.g.1,8-diamino-3,6-dioxaoctane or equivalents comprising longer ethyleneglycol chains), polyethylene glycol chains or polyethylene oxide chains,polypropylene glycol chains or polypropylene oxide chains and1,x-diaminoalkanes wherein x is the number of carbon atoms in thealkane. Another class of suitable spacer-moieties comprises cleavablespacer-moieties, or cleavable linkers. Cleavable linkers are well knownin the art. For example Shabat et al., Soft Matter 2012, 6, 1073,incorporated by reference herein, discloses cleavable linkers comprisingself-immolative moieties that are released upon a biological trigger,e.g. an enzymatic cleavage or an oxidation event. Some examples ofsuitable cleavable linkers are disulfide-linkers that are cleaved uponreduction, peptide-linkers that are cleaved upon specific recognition bya protease, e.g. cathepsin, plasmin or metalloproteases, orglycoside-based linkers that are cleaved upon specific recognition by aglycosidase, e.g. glucoronidase, or nitroaromatics that are reduced inoxygen-poor, hypoxic areas. Herein, suitable cleavable spacer-moietiesalso include spacer moieties comprising a specific, cleavable, sequenceof amino acids. Examples include e.g. spacer-moieties comprising aVal-Ala (valine-alanine) or Val-Cit (valine-citrulline) moiety.Bioconjugates containing a cleavable linker, such as Val-Cit linker, inparticular Val-Cit-PABC, suffer considerably from aggregation in view oftheir limited water-solubility. For such bioconjugates, incorporatingthe sulfamide linker according to the invention is particularlybeneficial. Also, conjugation reactions with a linker comprising acleavable linker are hampered by the limited water-solubility of thelinker. Hence, linker comprising a cleavable linker, such as Val-Citlinker, in particular Val-Cit-PABC, and the sulfamide linker accordingto the invention outperform linkers comprising such a cleavable linkerbut lacking such sulfamide linker in conjugation to biomolecules.

Thus, in a preferred embodiment of the linker according to formula (2),spacer moieties Sp¹, Sp², Sp³, Sp⁴, Sp⁵ and/or Sp⁶, if present, comprisea sequence of amino acids. Spacer-moieties comprising a sequence ofamino acids are known in the art, and may also be referred to as peptidelinkers. Examples include spacer-moieties comprising a Val-Cit moiety,e.g. Val-Cit-PABC, Val-Cit-PABC, Fmoc-Val-Cit-PABC, etc. Preferably, aVal-Cit-PABC moiety is employed in the linker.

In a preferred embodiment of the linker according to formula (2), spacermoieties Sp¹, Sp², Sp³, Sp⁴, Sp⁵ and Sp⁶, if present, are independentlyselected from the group consisting of linear or branched C₁-C₂₀₀alkylene groups, C₂-C₂₀₀ alkenylene groups, C₂-C₂₀₀ alkynylene groups,C₃-C₂₀₀ cycloalkylene groups, C₅-C₂₀₀ cycloalkenylene groups, C₈-C₂₀₀cycloalkynylene groups, C₇-C₂₀₀ alkylarylene groups, C₇-C₂₀₀arylalkylene groups, C₈-C₂₀₀ arylalkenylene groups and C₉-C₂₀₀arylalkynylene groups, the alkylene groups, alkenylene groups,alkynylene groups, cycloalkylene groups, cycloalkenylene groups,cycloalkynylene groups, alkylarylene groups, arylalkylene groups,arylalkenylene groups and arylalkynylene groups being optionallysubstituted and optionally interrupted by one or more heteroatomsselected from the group of O, S and NR³, wherein R³ is independentlyselected from the group consisting of hydrogen, C₁-C₂₄ alkyl groups,C₂-C₂₄ alkenyl groups, C₂-C₂₄ alkynyl groups and C₃-C₂₄ cycloalkylgroups, the alkyl groups, alkenyl groups, alkynyl groups and cycloalkylgroups being optionally substituted. When the alkylene groups,alkenylene groups, alkynylene groups, cycloalkylene groups,cycloalkenylene groups, cycloalkynylene groups, alkylarylene groups,arylalkylene groups, arylalkenylene groups and arylalkynylene groups areinterrupted by one or more heteroatoms as defined above, it is preferredthat said groups are interrupted by one or more O-atoms, and/or by oneor more S—S groups.

More preferably, spacer moieties Sp¹, Sp², Sp³, Sp⁴, Sp⁵ and Sp⁶, ifpresent, are independently selected from the group consisting of linearor branched C₁-C₁₀₀ alkylene groups, C₂-C₁₀₀ alkenylene groups, C₂-C₁₀₀alkynylene groups, C₃-C₁₀₀ cycloalkylene groups, C₅-C₁₀₀ cycloalkenylenegroups, C₅-C₁₀₀ cycloalkynylene groups, C₇-C₁₀₀ alkylarylene groups,C₇-C₁₀₀ arylalkylene groups, C₅-C₁₀₀ arylalkenylene groups and C₉-C₁₀₀arylalkynylene groups, the alkylene groups, alkenylene groups,alkynylene groups, cycloalkylene groups, cycloalkenylene groups,cycloalkynylene groups, alkylarylene groups, arylalkylene groups,arylalkenylene groups and arylalkynylene groups being optionallysubstituted and optionally interrupted by one or more heteroatomsselected from the group of O, S and NR³, wherein R³ is independentlyselected from the group consisting of hydrogen, C₁-C₂₄ alkyl groups,C₂-C₂₄ alkenyl groups, C₂-C₂₄ alkynyl groups and C₃-C₂₄ cycloalkylgroups, the alkyl groups, alkenyl groups, alkynyl groups and cycloalkylgroups being optionally substituted.

Even more preferably, spacer moieties Sp¹, Sp², Sp³, Sp⁴, Sp⁵ and Sp⁶,if present, are independently selected from the group consisting oflinear or branched C₁-C₅₀ alkylene groups, C₂-C₅₀ alkenylene groups,C₂-C₅₀ alkynylene groups, C₃-C₅₀ cycloalkylene groups, C₅-C₅₀cycloalkenylene groups, C₅-C₅₀ cycloalkynylene groups, C₇-C₅₀alkylarylene groups, C₇-C₅₀ arylalkylene groups, C₅-C₅₀ arylalkenylenegroups and C₉-C₅₀ arylalkynylene groups, the alkylene groups, alkenylenegroups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups,cycloalkynylene groups, alkylarylene groups, arylalkylene groups,arylalkenylene groups and arylalkynylene groups being optionallysubstituted and optionally interrupted by one or more heteroatomsselected from the group of O, S and NR³, wherein R³ is independentlyselected from the group consisting of hydrogen, C₁-C₂₄ alkyl groups,C₂-C₂₄ alkenyl groups, C₂-C₂₄ alkynyl groups and C₃-C₂₄ cycloalkylgroups, the alkyl groups, alkenyl groups, alkynyl groups and cycloalkylgroups being optionally substituted.

Yet even more preferably, spacer moieties Sp¹, Sp², Sp³, Sp⁴, Sp⁵ andSp⁶, if present, are independently selected from the group consisting oflinear or branched C₁-C₂₀ alkylene groups, C₂-C₂₀ alkenylene groups,C₂-C₂₀ alkynylene groups, C₃-C₂₀ cycloalkylene groups, C₅-C₂₀cycloalkenylene groups, C₈-C₂₀ cycloalkynylene groups, C₇-C₂₀alkylarylene groups, C₇-C₂₀ arylalkylene groups, C₈-C₂₀ arylalkenylenegroups and C₉-C₂₀ arylalkynylene groups, the alkylene groups, alkenylenegroups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups,cycloalkynylene groups, alkylarylene groups, arylalkylene groups,arylalkenylene groups and arylalkynylene groups being optionallysubstituted and optionally interrupted by one or more heteroatomsselected from the group of O, S and NR³, wherein R³ is independentlyselected from the group consisting of hydrogen, C₁-C₂₄ alkyl groups,C₂-C₂₄ alkenyl groups, C₂-C₂₄ alkynyl groups and C₃-C₂₄ cycloalkylgroups, the alkyl groups, alkenyl groups, alkynyl groups and cycloalkylgroups being optionally substituted.

In these preferred embodiments it is further preferred that the alkylenegroups, alkenylene groups, alkynylene groups, cycloalkylene groups,cycloalkenylene groups, cycloalkynylene groups, alkylarylene groups,arylalkylene groups, arylalkenylene groups and arylalkynylene groups areunsubstituted and optionally interrupted by one or more heteroatomsselected from the group of O, S and NR³, preferably 0, wherein R³ isindependently selected from the group consisting of hydrogen and C₁-C₄alkyl groups, preferably hydrogen or methyl.

Most preferably, spacer moieties Sp¹, Sp², Sp³, Sp⁴, Sp⁵ and Sp⁶, ifpresent, are independently selected from the group consisting of linearor branched C₁-C₂₀ alkylene groups, the alkylene groups being optionallysubstituted and optionally interrupted by one or more heteroatomsselected from the group of O, S and NR³, wherein R³ is independentlyselected from the group consisting of hydrogen, C₁-C₂₄ alkyl groups,C₂-C₂₄ alkenyl groups, C₂-C₂₄ alkynyl groups and C₃-C₂₄ cycloalkylgroups, the alkyl groups, alkenyl groups, alkynyl groups and cycloalkylgroups being optionally substituted. In this embodiment, it is furtherpreferred that the alkylene groups are unsubstituted and optionallyinterrupted by one or more heteroatoms selected from the group of O, Sand NR³, preferably O and/or or S—S, wherein R³ is independentlyselected from the group consisting of hydrogen and C₁-C₄ alkyl groups,preferably hydrogen or methyl.

Preferred spacer moieties Sp¹, Sp², Sp³, Sp⁴, Sp⁵ and Sp⁶ thus include—(CH₂)_(n)—, —(CH₂CH₂)_(n)—, —(CH₂CH₂O)_(n)—, —(OCH₂CH₂)_(n)—,—(CH₂CH₂O)_(n)CH₂CH₂—, —CH₂CH₂(OCH₂CH₂)_(n)—, —(CH₂CH₂CH₂O)_(n)—,—(OCH₂CH₂CH₂)_(n)—, —(CH₂CH₂CH₂O)_(n)CH₂CH₂CH₂— and—CH₂CH₂CH₂(OCH₂CH₂CH₂)_(n)—, wherein n is an integer in the range of 1to 50, preferably in the range of 1 to 40, more preferably in the rangeof 1 to 30, even more preferably in the range of 1 to 20 and yet evenmore preferably in the range of 1 to 15. More preferably n is 1, 2, 3,4, 5, 6, 7, 8, 9 or 10, more preferably 1, 2, 3, 4, 5, 6, 7 or 8, evenmore preferably 1, 2, 3, 4, 5 or 6, yet even more preferably 1, 2, 3 or4.

Since Sp¹, Sp², Sp³, Sp⁴, Sp⁵ and Sp⁶ are independently selected, Sp¹,if present, may be different from Sp², if present, which may bedifferent from Sp³, if present, which may be different from Sp⁴, ifpresent, which may be different from Sp⁵, if present, which may bedifferent from Sp⁶, if present.

As described above, R¹ is selected from the group consisting ofhydrogen, C₁-C₂₄ alkyl groups, C₃-C₂₄ cycloalkyl groups, C₂-C₂₄(hetero)aryl groups, C₃-C₂₄ alkyl(hetero)aryl groups and C₃-C₂₄(hetero)arylalkyl groups, the C₁-C₂₄ alkyl groups, C₃-C₂₄ cycloalkylgroups, C₂-C₂₄ (hetero)aryl groups, C₃-C₂₄ alkyl(hetero)aryl groups andC₃-C₂₄ (hetero)arylalkyl groups optionally substituted and optionallyinterrupted by one or more heteroatoms selected from O, S and NR³wherein R³ is independently selected from the group consisting ofhydrogen and C₁-C₄ alkyl groups, or R¹ is D,-[(Sp¹)_(b)—(Z²)_(e)—(Sp⁴)_(i)-D] or -[(Sp²)_(c)—(Z¹)_(d)—(Sp³)_(g)-Q¹],wherein Sp¹, Sp², Sp³, Sp⁴, Z¹, Z², D, Q¹, b, c, d, e, g and i are asdefined above.

In a preferred embodiment, R¹ is hydrogen or a C₁-C₂₀ alkyl group, morepreferably R¹ is hydrogen or a C₁-C₁₆ alkyl group, even more preferablyR¹ is hydrogen or a C₁-C₁ alkyl group, wherein the alkyl group isoptionally substituted and optionally interrupted by one or moreheteroatoms selected from O, S and NR³, preferably 0, wherein R³ isindependently selected from the group consisting of hydrogen and C₁-C₄alkyl groups. In a further preferred embodiment, R¹ is hydrogen. Inanother further preferred embodiment, R¹ is a C₁-C₂₀ alkyl group, morepreferably a C₁-C₁₆ alkyl group, even more preferably a C₁-C₁ alkylgroup, wherein the alkyl group is optionally interrupted by one or moreO-atoms, and wherein the alkyl group is optionally substituted with an—OH group, preferably a terminal —OH group. In this embodiment it isfurther preferred that R¹ is a polyethylene glycol chain comprising aterminal —OH group. In another further preferred embodiment, R¹ is aC₁-C₁₂ alkyl group, more preferably a C₁-C₆ alkyl group, even morepreferably a C₁-C₄ alkyl group, and yet even more preferably R¹ isselected from the group consisting of methyl, ethyl, n-propyl, i-propyl,n-butyl, s-butyl and t-butyl. In another preferred embodiment, R¹ is atarget molecule D, -[(Sp¹)_(b)—(Z²)_(e)—(Sp⁴)_(i)-D] or-[(Sp²)_(c)—(Z¹)_(d)—(Sp³)_(g)-Q¹], wherein Sp¹, Sp², Sp³, Sp⁴, Z¹, Z²,D, Q¹, b, c, d, e, g and i are as defined above.

In the linker according to formula (2), f is an integer in the range of1 to 10, preferably 1-5. The linker may thus comprise more than onegroup according to formula (1), the group according to formula (1) beingas defined above. When more than one group according to formula (1) ispresent, i.e. When f is 2 or more, then BM, b, Sp¹, Sp⁵, k, SG Sp⁶, Iand E are independently selected. In other words, when f is 2 or more,each b is independently 0 or 1, each k is independently 0 or 1, each Iis independently 0 or 1, each Sp¹ may be the same or different, each Sp⁵may be the same or different, each Sp⁶ may be the same or different eachSG may be the same or different and each E may be the same or different.f is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably f is 1, 2, 3, 4, or 5,even more preferably f is 1, 2, 3 or 4, even more preferably f is 1, 2or 3, even more preferably f is 1 or 2, and most preferably f is 1 inthis embodiment. In another preferred embodiment, f is an integer in therange of 2 to 10, i.e. f is 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably fis 2, 3, 4 or 5, more preferably f is 2, 3 or 4, even more preferably fis 2 or 3, and most preferably f is 2 in this embodiment.

These preferred embodiments of the linker of structure (2) also hold forthe linker-moiety in linker-conjugates, linker-constructs andbioconjugates according to the invention as described in more detailbelow.

Linker-Conjugate

In the context of the present invention, “linker-conjugate” refers tothe target molecule D which is functionalized with a linker according tothe invention, comprising a branching moiety BM and a sulfamide groupSG, which further bears a reactive group Q¹. In other words, thelinker-conjugate is a compound wherein a target molecule is covalentlyconnected to a reactive group Q¹, via a linker. In a first aspect, theinvention concerns linker-conjugates as defined herein. Thelinker-conjugate according to the invention comprises the linkeraccording to the invention as defined above, preferably the linkeraccording to structure (2).

The linker-conjugate according to the invention may comprise more thanone target molecule D. Similarly, the linker-conjugate may comprise morethan one reactive group Q¹. When more than one reactive group Q¹ ispresent the groups Q¹ may be the same or different, and when more thanone target molecule D is present the target molecules D may be the sameor different. The linker-conjugate according to the invention maytherefore also be denoted as (Q¹)_(y)-Sp-(D)_(z), wherein y is aninteger in the range of 1 to 10 and z is an integer in the range of 1 to10. The invention thus also relates to a compound according to theformula: (Q¹)_(y)-Sp-(D)_(z), wherein:

-   -   y is an integer in the range of 1 to 10, preferably in the range        1-5, more preferably y=1;    -   z is an integer in the range of 1 to 10, preferably in the range        1-5;    -   Q¹ is a reactive group capable of reacting with a functional        group F¹ present on a biomolecule;    -   D is a target molecule;    -   Sp is a spacer moiety, wherein a spacer moiety is defined as a        moiety that spaces (i.e. provides a certain distance between)        and covalently links reactive group Q¹ and target molecule D,        wherein said spacer moiety comprises the linker according to the        invention as defined above.

Preferably, y is 1, 2, 3 or 4, more preferably y is 1 or 2 and mostpreferably, y is 1. Preferably, z is 1, 2, 3, 4, 5 or 6, more preferablyz is 1, 2, 3 or 4, even more preferably z is 1, 2 or 3, yet even morepreferably z is 1 or 2 and most preferably z is 1. More preferably, y is1 or 2, preferably 1, and z is 1, 2, 3 or 4, yet even more preferably yis 1 or 2, preferably 1, and z is 1, 2 or 3, yet even more preferably yis 1 or 2, preferably 1, and z is 1 or 2, and most preferably y is 1 andz is 1. In a preferred embodiment, the linker-conjugate is according tothe formula Q¹-Sp-(D)₄, Q¹-Sp-(D)₃, Q¹-Sp-(D)₂ or Q¹-Sp-D.

Target Molecule D

Target molecules in the field of bioconjugation are known to the skilledperson, and may also be referred to as payload. In a preferredembodiment of the linker-conjugate according to the invention, thetarget molecule is selected from the group consisting of an activesubstance, a reporter molecule, a polymer, a solid surface, a hydrogel,a nanoparticle, a microparticle and a biomolecule.

The inventors found that the use of a sulfamide linker according to theinvention improves the solubility of the linker-conjugate, which in turnsignificantly improves the efficiency of the conjugation. Usingconventional linkers, effective conjugation is often hampered by therelatively low solubility of the linker-conjugate in aqueous media,especially when a relative water-insoluble or hydrophobic targetmolecule is used. In a particularly preferred embodiment, the targetmolecule in its unconjugated form is hydrophobic, typically having awater solubility of at most 1% (w/w), preferably at most 0.1% (w/w),most preferably at most 0.01% (w/w), determined at 20° C. and 100 kPa.Even such water-insoluble target molecules are effectively subjected toconjugation when functionalized with a sulfamide linker according to theinvention. Herein, the “unconjugated form” refers to the target moleculenot being functionalized with or conjugated to the linker according tothe invention. Such unconjugated forms of target molecules are known tothe skilled person.

The term “active substance” herein relates to a pharmacological and/orbiological substance, i.e. a substance that is biologically and/orpharmaceutically active, for example a drug, a prodrug, a diagnosticagent, a protein, a peptide, a polypeptide, a peptide tag, an aminoacid, a glycan, a lipid, a vitamin, a steroid, a nucleotide, anucleoside, a polynucleotide, RNA or DNA. Examples of peptide tagsinclude cell-penetrating peptides like human lactoferrin orpolyarginine. An example of a glycan is oligomannose. An example of anamino acid is lysine.

When the target molecule is an active substance, the active substance ispreferably selected from the group consisting of drugs and prodrugs.More preferably, the active substance is selected from the groupconsisting of pharmaceutically active compounds, in particular low tomedium molecular weight compounds (e.g. about 200 to about 2500 Da,preferably about 300 to about 1750 Da). In a further preferredembodiment, the active substance is selected from the group consistingof cytotoxins, antiviral agents, antibacterials agents, peptides andoligonucleotides. Examples of cytotoxins include colchicine, vincaalkaloids, anthracyclines, camptothecins, doxorubicin, daunorubicin,taxanes, calicheamycins, tubulysins, irinotecans, an inhibitory peptide,amanitin, deBouganin, duocarmycins, maytansines, auristatins orpyrrolobenzodiazepines (PBDs). In view of their poor water solubility,preferred active substances include vinca alkaloids, anthracyclines,camptothecins, taxanes, tubulysins, amanitin, duocarmycins, maytansines,auristatins and pyrrolobenzodiazepines, in particular vinca alkaloids,anthracyclines, camptothecins, taxanes, tubulysins, amanitin,maytansines and auristatins.

The term “reporter molecule” herein refers to a molecule whose presenceis readily detected, for example a diagnostic agent, a dye, afluorophore, a radioactive isotope label, a contrast agent, a magneticresonance imaging agent or a mass label.

A wide variety of fluorophores, also referred to as fluorescent probes,is known to a person skilled in the art. Several fluorophores aredescribed in more detail in e.g. G. T. Hermanson, “BioconjugateTechniques”, Elsevier, 3^(rd) Ed. 2013, Chapter 10: “Fluorescentprobes”, p. 395-463, incorporated by reference. Examples of afluorophore include all kinds of Alexa Fluor (e.g. Alexa Fluor 555),cyanine dyes (e.g. Cy3 or Cy5) and cyanine dye derivatives, coumarinderivatives, fluorescein and fluorescein derivatives, rhodamine andrhodamine derivatives, boron dipyrromethene derivatives, pyrenederivatives, naphthalimide derivatives, phycobiliprotein derivatives(e.g. allophycocyanin), chromomycin, lanthanide chelates and quantum dotnanocrystals. In view of their poor water solubility, preferredfluorophores include cyanine dyes, coumarin derivatives, fluorescein andderivatives thereof, pyrene derivatives, naphthalimide derivatives,chromomycin, lanthanide chelates and quantum dot nanocrystals, inparticular coumarin derivatives, fluorescein, pyrene derivatives andchromomycin.

Examples of a radioactive isotope label include ^(99m)Tc, ¹¹¹In, ¹¹⁴In,¹¹⁵In, ¹⁸F, ¹⁴C, ⁶⁴Cu, ¹³¹I, ¹²⁵I, ¹²³I, ²¹²Bi, ⁸⁸Y, ⁹⁰Y, ⁶⁷Cu, ¹⁸⁶Rh,¹⁸⁸Rh, ⁶⁶Ga, ⁶⁷Ga and ¹⁰B, which is optionally connected via a chelatingmoiety such as e.g. DTPA (diethylenetriaminepentaacetic anhydride), DOTA(1,4,7,10-tetraazacyclododecane-N,N′N″,N′″-tetraacetic acid), NOTA(1,4,7-triazacyclononane N,N′,N″-triacetic acid), TETA(1,4,8,11-tetraazacyclotetradecane-N,N′,N″,N′″tetraacetic acid), DTTA(N¹-(p-isothiocyanatobenzyl)-diethylenetriamine-N¹,N²,N³,N³-tetraaceticacid), deferoxamine or DFA(N′-[5-[[4-[[5-(acetylhydroxyamino)pentyl]amino]-1,4-dioxobutyl]hydroxyamino]pentyl]-N-(5-aminopentyl)-N-hydroxybutanediamide)or HYNIC (hydrazinonicotinamide). Isotopic labelling techniques areknown to a person skilled in the art, and are described in more detailin e.g. G. T. Hermanson, “Bioconjugate Techniques”, Elsevier, 3^(rd) Ed.2013, Chapter 12: “Isotopic labelling techniques”, p. 507-534,incorporated by reference.

Polymers suitable for use as a target molecule D in the compoundaccording to the invention are known to a person skilled in the art, andseveral examples are described in more detail in e.g. G. T. Hermanson,“Bioconjugate Techniques”, Elsevier, 3^(rd) Ed. 2013, Chapter 18:“PEGylation and synthetic polymer modification”, p. 787-838,incorporated by reference. When target molecule D is a polymer, targetmolecule D is preferably independently selected from the groupconsisting of a poly(ethylene glycol) (PEG), a polyethylene oxide (PEO),a polypropylene glycol (PPG), a polypropylene oxide (PPO), a1,x-diaminoalkane polymer (wherein x is the number of carbon atoms inthe alkane, and preferably x is an integer in the range of 2 to 200,preferably 2 to 10), a (poly)ethylene glycol diamine (e.g.1,8-diamino-3,6-dioxaoctane and equivalents comprising longer ethyleneglycol chains), a polysaccharide (e.g. dextran), a poly(amino acid)(e.g. a poly(L-lysine)) and a poly(vinyl alcohol). In view of their poorwater solubility, preferred polymers include a 1,x-diaminoalkane polymerand poly(vinyl alcohol).

Solid surfaces suitable for use as a target molecule D are known to aperson skilled in the art. A solid surface is for example a functionalsurface (e.g. a surface of a nanomaterial, a carbon nanotube, afullerene or a virus capsid), a metal surface (e.g. a titanium, gold,silver, copper, nickel, tin, rhodium or zinc surface), a metal alloysurface (wherein the alloy is from e.g. aluminium, bismuth, chromium,cobalt, copper, gallium, gold, indium, iron, lead, magnesium, mercury,nickel, potassium, plutonium, rhodium, scandium, silver, sodium,titanium, tin, uranium, zinc and/or zirconium), a polymer surface(wherein the polymer is e.g. polystyrene, polyvinylchloride,polyethylene, polypropylene, poly(dimethylsiloxane) orpoly(methylmethacrylate), poly(acrylamide)), a glass surface, a siliconesurface, a chromatography support surface (wherein the chromatographysupport is e.g. a silica support, an agarose support, a cellulosesupport or an alumina support), etc. When target molecule D is a solidsurface, it is preferred that D is independently selected from the groupconsisting of a functional surface or a polymer surface.

Hydrogels are known to the person skilled in the art. Hydrogels arewater-swollen networks, formed by cross-links between the polymericconstituents. See for example A. S. Hoffman, Adv. Drug Delivery Rev.2012, 64, 18, incorporated by reference. When the target molecule is ahydrogel, it is preferred that the hydrogel is composed ofpoly(ethylene)glycol (PEG) as the polymeric basis.

Micro- and nanoparticles suitable for use as a target molecule D areknown to a person skilled in the art. A variety of suitable micro- andnanoparticles is described in e.g. G. T. Hermanson, “BioconjugateTechniques”, Elsevier, 3^(rd) Ed. 2013, Chapter 14: “Microparticles andnanoparticles”, p. 549-587, incorporated by reference. The micro- ornanoparticles may be of any shape, e.g. spheres, rods, tubes, cubes,triangles and cones. Preferably, the micro- or nanoparticles are of aspherical shape. The chemical composition of the micro- andnanoparticles may vary. When target molecule D is a micro- or ananoparticle, the micro- or nanoparticle is for example a polymericmicro- or nanoparticle, a silica micro- or nanoparticle or a gold micro-or nanoparticle. When the particle is a polymeric micro- ornanoparticle, the polymer is preferably polystyrene or a copolymer ofstyrene (e.g. a copolymer of styrene and divinylbenzene, butadiene,acrylate and/or vinyltoluene), poly(methylmethacrylate) (PMMA),poly(vinyltoluene), poly(hydroxyethyl methacrylate) p(HEMA) orpoly(ethylene glycol dimethacrylate/2-hydroxyethylmethacrylate)p(EDGMA/HEMA). Optionally, the surface of the micro- or nanoparticles ismodified, e.g. with detergents, by graft polymerization of secondarypolymers or by covalent attachment of another polymer or of spacermoieties, etc.

Target molecule D may also be a biomolecule. Biomolecules, and preferredembodiments thereof, are described in more detail below. When targetmolecule D is a biomolecule, it is preferred that the biomolecule isselected from the group consisting of proteins (including glycoproteinsand antibodies), polypeptides, peptides, glycans, lipids, nucleic acids,oligonucleotides, polysaccharides, oligosaccharides, enzymes, hormones,amino acids and monosaccharides.

Reactive Moiety Q¹

The linker-conjugate according to the invention comprises a reactivegroup Q¹ that is capable of reacting with a functional group F¹ presenton a biomolecule. Functional groups are known to a person skilled in theart and may be defined as any molecular entity that imparts a specificproperty onto the molecule harbouring it. For example, a functionalgroup in a biomolecule may constitute an amino group, a thiol group, acarboxylic acid, an alcohol group, a carbonyl group, a phosphate group,or an aromatic group. The functional group in the biomolecule may benaturally present or may be placed in the biomolecule by a specifictechnique, for example a (bio)chemical or a genetic technique. Thefunctional group that is placed in the biomolecule may be a functionalgroup that is naturally present in nature, or may be a functional groupthat is prepared by chemical synthesis, for example an azide, a terminalalkyne or a phosphine moiety. Herein, the term “reactive group” mayrefer to a certain group that comprises a functional group, but also toa functional group itself. For example, a cyclooctynyl group is areactive group comprising a functional group, namely a C—C triple bond.Similarly, an N-maleimidyl group is a reactive group, comprising a C—Cdouble bond as a functional group. However, a functional group, forexample an azido functional group, a thiol functional group or an aminofunctional group, may herein also be referred to as a reactive group.

The linker-conjugate may comprise more than one reactive group Q¹. Whenthe linker-conjugate comprises two or more reactive groups Q¹, thereactive groups Q¹ may differ from each other. Preferably, thelinker-conjugate comprises one reactive group Q¹.

Reactive group Q¹ that is present in the linker-conjugate, is able toreact with a functional group F¹ that is present in a biomolecule. Inother words, reactive group Q¹ needs to be complementary to a functionalgroup F¹ present in a biomolecule. Herein, a reactive group is denotedas “complementary” to a functional group when said reactive group reactswith said functional group selectively, optionally in the presence ofother functional groups. Complementary reactive and functional groupsare known to a person skilled in the art, and are described in moredetail below. Preferably, reactive group Q¹ and functional group F1 arecapable of reacting in a bioorthogonal reaction, as those reactions donot interfere with the biomolecules present during this reaction.Bioorthogonal reactions and functional groups suitable therein are knownto the skilled person, for example from Gong and Pan, Tetrahedron Lett.2015, 56, 2123-2132, and include Staudinger ligations and copper-freeClick chemistry. It is thus preferred that Q¹ is selected from the groupconsisting of 1,3-dipoles, alkynes, (hetero)cyclooctynes, cyclooctenes,tetrazines, ketones, aldehydes, alkoxyamines, hydrazines andtriphenylphosphine. In a preferred embodiment, reactive group Q¹ isselected from the group consisting of, optionally substituted,N-maleimidyl groups, halogenated N-alkylamido groups, sulfonyloxyN-alkylamido groups, ester groups, carbonate groups, sulfonyl halidegroups, thiol groups or derivatives thereof, alkenyl groups, alkynylgroups, (hetero)cycloalkynyl groups, bicyclo[6.1.0]non-4-yn-9-yl]groups, cycloalkenyl groups, tetrazinyl groups, azido groups, phosphinegroups, nitrile oxide groups, nitrone groups, nitrile imine groups,diazo groups, ketone groups, (O-alkyl)hydroxylamino groups, hydrazinegroups, halogenated N-maleimidyl groups,1,1-bis(sulfonylmethyl)methylcarbonyl groups or elimination derivativesthereof, carbonyl halide groups, allenamide groups, 1,2-quinone groupsor triazine groups.

In a preferred embodiment, Q¹ is an N-maleimidyl group. When Q¹ is anN-maleimidyl group, Q¹ is preferably unsubstituted. Q¹ is thuspreferably according to formula (9a), as shown below. Suitablemaleimidyl moieties include diaminopropionyl maleimdyl moieties. Apreferred example of such a maleimidyl group is 2,3-diaminopropionicacid (DPR) maleimidyl, which may be connected to the remainder of thelinker-conjugate through the carboxylic acid moiety or the remainingamine moiety.

In another preferred embodiment, Q¹ is a halogenated N-alkylamido group.When Q¹ is a halogenated N-alkylamido group, it is preferred that Q¹ isaccording to formula (9b), as shown below, wherein k is an integer inthe range of 1 to 10 and R⁴ is selected from the group consisting of—Cl, —Br and —I. Preferably k is 1, 2, 3 or 4, more preferably k is 1 or2 and most preferably k is 1. Preferably, R⁴ is —I or —Br. Morepreferably, k is 1 or 2 and R⁴ is —I or —Br, and most preferably k is 1and R⁴ is —I or Br.

In another preferred embodiment, Q¹ is a sulfonyloxy N-alkylamido group.When Q¹ is a sulfonyloxy N-alkylamido group, it is preferred that Q¹ isaccording to formula (9b), as shown below, wherein k is an integer inthe range of 1 to 10 and R⁴ is selected from the group consisting of—O-mesyl, —O-phenylsulfonyl and —O-tosyl. Preferably k is 1, 2, 3 or 4,more preferably k is 1 or 2, even more preferably k is 1. Mostpreferably k is 1 and R⁴ is selected from the group consisting of—O-mesyl, —O-phenylsulfonyl and —O-tosyl.

In another preferred embodiment, Q¹ is an ester group. When Q¹ is anester group, it is preferred that the ester group is an activated estergroup. Activated ester groups are known to the person skilled in theart. An activated ester group is herein defined as an ester groupcomprising a good leaving group, wherein the ester carbonyl group isbonded to said good leaving group. Good leaving groups are known to theperson skilled in the art. It is further preferred that the activatedester is according to formula (9c), as shown below, wherein R⁵ isselected from the group consisting of —N-succinimidyl (NHS),—N-sulfo-succinimidyl (sulfo-NHS), -(4-nitrophenyl), -pentafluorophenylor -tetrafluorophenyl (TFP).

In another preferred embodiment, Q¹ is a carbonate group. When Q¹ is acarbonate group, it is preferred that the carbonate group is anactivated carbonate group. Activated carbonate groups are known to aperson skilled in the art. An activated carbonate group is hereindefined as a carbonate group comprising a good leaving group, whereinthe carbonate carbonyl group is bonded to said good leaving group. It isfurther preferred that the carbonate group is according to formula (9d),as shown below, wherein R⁷ is selected from the group consisting of —N—succinimidyl, —N-sulfo-succinimidyl, -(4-nitrophenyl),-pentafluorophenyl or -tetrafluorophenyl.

In another preferred embodiment, Q¹ is a sulfonyl halide group accordingto formula (9e) as shown below, wherein X is selected from the groupconsisting of F, Cl, Br and I. Preferably X is C₁ or Br, more preferablyCl.

In another preferred embodiment, Q¹ is a thiol group (9f), or aderivative or a precursor of a thiol group. A thiol group may also bereferred to as a mercapto group. When Q¹ is a derivative or a precursorof a thiol group, the thiol derivative is preferably according toformula (9g), (9h) or (9zb) as shown below, wherein R⁸ is an, optionallysubstituted, C₁-C₁₂ alkyl group or a C₂-C₁₂ (hetero)aryl group, V is Oor S and R¹⁶ is an, optionally substituted, C₁-C₁₂ alkyl group. Morepreferably R⁸ is an, optionally substituted, C₁-C₆ alkyl group or aC₂-C₆ (hetero)aryl group, and even more preferably R⁸ is methyl, ethyl,n-propyl, i-propyl, n-butyl, s-butyl, t-butyl or phenyl. Even morepreferably, R⁸ is methyl or phenyl, most preferably methyl. Morepreferably R¹⁶ is an optionally substituted C₁-C₆ alkyl group, and evenmore preferably R¹⁶ is methyl, ethyl, n-propyl, i-propyl, n-butyl,s-butyl or t-butyl, most preferably methyl. When Q¹ is athiol-derivative according to formula (9g) or (9zb), and Q¹ is reactedwith a reactive group F¹ on a biomolecule, said thiol-derivative needsto be converted into a thiol group during the process. When Q¹ isaccording to formula (9h), Q¹ is —SC(O)OR⁸ or —SC(S)OR⁸, preferablySC(O)OR⁸, wherein R⁸, and preferred embodiments thereof, are as definedabove.

In another preferred embodiment, Q¹ is an alkenyl group, wherein thealkenyl group is linear or branched, and wherein the alkenyl group isoptionally substituted. The alkenyl group may be a terminal or aninternal alkenyl group. The alkenyl group may comprise more than one C—Cdouble bond, and if so, preferably comprises two C—C double bonds. Whenthe alkenyl group is a dienyl group, it is further preferred that thetwo C—C double bonds are separated by one C—C single bond (i.e. it ispreferred that the dienyl group is a conjugated dienyl group).Preferably said alkenyl group is a C₂-C₂₄ alkenyl group, more preferablya C₂-C₁₂ alkenyl group, and even more preferably a C₂-C₆ alkenyl group.It is further preferred that the alkenyl group is a terminal alkenylgroup. More preferably, the alkenyl group is according to formula (9i)as shown below, wherein l is an integer in the range of 0 to 10,preferably in the range of 0 to 6, and p is an integer in the range of 0to 10, preferably 0 to 6. More preferably, l is 0, 1, 2, 3 or 4, morepreferably l is 0, 1 or 2 and most preferably l is 0 or 1. Morepreferably, p is 0, 1, 2, 3 or 4, more preferably p is 0, 1 or 2 andmost preferably p is 0 or 1. It is particularly preferred that p is 0and l is 0 or 1, or that p is 1 and l is 0 or 1.

In another preferred embodiment, Q¹ is an alkynyl group, wherein thealkynyl group is linear or branched, and wherein the alkynyl group isoptionally substituted. The alkynyl group may be a terminal or aninternal alkynyl group. Preferably said alkynyl group is a C₂-C₂₄alkynyl group, more preferably a C₂-C₁₂ alkynyl group, and even morepreferably a C₂-C₆ alkynyl group. It is further preferred that thealkynyl group is a terminal alkynyl group. More preferably, the alkynylgroup is according to formula (9j) as shown below, wherein l is aninteger in the range of 0 to 10, preferably in the range of 0 to 6. Morepreferably, l is 0, 1, 2, 3 or 4, more preferably l is 0, 1 or 2 andmost preferably l is 0 or 1.

In another preferred embodiment, Q¹ is a cycloalkenyl group. Thecycloalkenyl group is optionally substituted. Preferably saidcycloalkenyl group is a C₃-C₂₄ cycloalkenyl group, more preferably aC₃-C₁₂ cycloalkenyl group, and even more preferably a C₃-C₈ cycloalkenylgroup. In a preferred embodiment, the cycloalkenyl group is atrans-cycloalkenyl group, more preferably a trans-cyclooctenyl group(also referred to as a TCO group) and most preferably atrans-cyclooctenyl group according to formula (9zi) or (9zj) as shownbelow. In another preferred embodiment, the cycloalkenyl group is acyclopropenyl group, wherein the cyclopropenyl group is optionallysubstituted. In another preferred embodiment, the cycloalkenyl group isa norbornenyl group, an oxanorbornenyl group, a norbornadienyl group oran oxanorbornadienyl group, wherein the norbornenyl group,oxanorbornenyl group, norbornadienyl group or an oxanorbornadienyl groupis optionally substituted. In a further preferred embodiment, thecycloalkenyl group is according to formula (9k), (9l), (9m) or (9zc) asshown below, wherein T is CH₂ or O, R⁹ is independently selected fromthe group consisting of hydrogen, a linear or branched C₁-C₁₂ alkylgroup or a C₄-C₁₂ (hetero)aryl group, and R¹⁹ is selected from the groupconsisting of hydrogen and fluorinated hydrocarbons. Preferably, R⁹ isindependently hydrogen or a C₁-C₆ alkyl group, more preferably R⁹ isindependently hydrogen or a C₁-C₄ alkyl group. Even more preferably R⁹is independently hydrogen or methyl, ethyl, n-propyl, i-propyl, n-butyl,s-butyl or t-butyl. Yet even more preferably R⁹ is independentlyhydrogen or methyl. In a further preferred embodiment, R¹⁹ is selectedfrom the group of hydrogen and —CF₃, —C₂F₅, —C₃F₇ and —C₄F₉, morepreferably hydrogen and —CF₃. In a further preferred embodiment, thecycloalkenyl group is according to formula (9k), wherein one R⁹ ishydrogen and the other R₉ is a methyl group. In another furtherpreferred embodiment, the cycloalkenyl group is according to formula(9l), wherein both R⁹ are hydrogen. In these embodiments it is furtherpreferred that l is 0 or 1. In another further preferred embodiment, thecycloalkenyl group is a norbornenyl (T is CH₂) or an oxanorbornenyl (Tis O) group according to formula (9m), or a norbornadienyl (T is CH₂) oran oxanorbornadienyl (T is O) group according to formula (9zc), whereinR⁹ is hydrogen and R¹⁹ is hydrogen or —CF₃, preferably —CF₃.

In another preferred embodiment, Q¹ is a (hetero)cycloalkynyl group. The(hetero)cycloalkynyl group is optionally substituted. Preferably, the(hetero)cycloalkynyl group is a (hetero)cyclooctynyl group, i.e. aheterocyclooctynyl group or a cyclooctynyl group, wherein the(hetero)cyclooctynyl group is optionally substituted. In a furtherpreferred embodiment, the (hetero)cyclooctynyl group is according toformula (9n), also referred to as a DIBO group, (9o), also referred toas a DIBAC group or (9p), also referred to as a BARAC group, or (9zk),also referred to as a COMBO group, all as shown below, wherein U is O orNR⁹, and preferred embodiments of R⁹ are as defined above. The aromaticrings in (9n) are optionally O-sulfonylated at one or more positions,whereas the rings of (9o) and (9p) may be halogenated at one or morepositions.

In an especially preferred embodiment, the nitrogen atom attached to R¹in compound (4b) is the nitrogen atom in the ring of theheterocycloalkyne group such as the nitrogen atom in (9o). In otherwords, c, d and g are 0 in compound (4b) and R¹ and Q¹, together withthe nitrogen atom they are attached to, form a heterocycloalkyne group,preferably a heterocyclooctyne group, most preferably theheterocyclooctyne group according to formula (9o) or (9p). Herein, thecarbonyl moiety of (9o) is replaced by the sulfonyl group of the groupaccording to formula (1). Alternatively, the nitrogen atom to which R¹is attached is the same atom as the atom designated as U in formula(9n). In other words, when Q¹ is according to formula (9n), U may be theright nitrogen atom of the group according to formula (1), or U═NR⁹ andR⁹ is the remainder of the group according to formula (1) and R¹ is thecyclooctyne moiety.

In another preferred embodiment, Q¹ is an, optionally substituted,bicyclo[6.1.0]non-4-yn-9-yl]group, also referred to as a BCN group.Preferably, the bicyclo[6.1.0]non-4-yn-9-yl] group is according toformula (9q) as shown below.

In another preferred embodiment, Q¹ is a conjugated (hetero)diene groupcapable of reacting in a Diels-Alder reaction. Preferred (hetero)dienegroups include optionally substituted tetrazinyl groups, optionallysubstituted 1,2-quinone groups and optionally substituted triazinegroups. More preferably, said tetrazinyl group is according to formula(9r), as shown below, wherein R⁹ is selected from the group consistingof hydrogen, a linear or branched C₁-C₁₂ alkyl group or a C₄-C₁₂(hetero)aryl group. Preferably, R⁹ is hydrogen, a C₁-C₆ alkyl group or aC₄-C₁₀ (hetero)aryl group, more preferably R⁹ is hydrogen, a C₁-C₄ alkylgroup or a C₄-C₆ (hetero)aryl group. Even more preferably R⁹ ishydrogen, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butylor pyridyl. Yet even more preferably R⁹ is hydrogen, methyl or pyridyl.More preferably, said 1,2-quinone group is according to formula (9zl) or(9zm). Said triazine group may be any regioisomer. More preferably, saidtriazine group is a 1,2,3-triazine group or a 1,2,4-triazine group,which may be attached via any possible location, such as indicated informula (9zn). The 1,2,3-triazine is most preferred as triazine group.

In another preferred embodiment, Q¹ is an azido group according toformula (9s) as shown below. In another preferred embodiment, Q¹ is an,optionally substituted, triarylphosphine group that is suitable toundergo a Staudinger ligation reaction. Preferably, the phosphine groupis according to formula (9t) as shown below, wherein R¹⁰ is a(thio)ester group. When R¹⁰ is a (thio)ester group, it is preferred thatR¹⁰ is —C(O)—V—R¹¹, wherein V is O or S and R¹¹ is a C₁-C₁₂ alkyl group.Preferably, R¹¹ is a C₁-C₆ alkyl group, more preferably a C₁-C₄ alkylgroup. Most preferably, R¹¹ is a methyl group.

In another preferred embodiment, Q¹ is a nitrile oxide group accordingto formula (9u) as shown below.

In another preferred embodiment, Q¹ is a nitrone group. Preferably, thenitrone group is according to formula (9v) as shown below, wherein R¹²is selected from the group consisting of linear or branched C₁-C₁₂ alkylgroups and C₆-C₁₂ aryl groups. Preferably, R¹² is a C₁-C₆ alkyl group,more preferably R¹² is a C₁-C₄ alkyl group. Even more preferably R¹² ismethyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl or t-butyl. Yet evenmore preferably R¹² is methyl.

In another preferred embodiment, Q¹ is a nitrile imine group.Preferably, the nitrile imine group is according to formula (9w) or(9zd) as shown below, wherein R¹³ is selected from the group consistingof linear or branched C₁-C₁₂ alkyl groups and C₆-C₁₂ aryl groups.Preferably, R¹³ is a C₁-C₆ alkyl group, more preferably R¹³ is a C₁-C₄alkyl group. Even more preferably R¹³ is methyl, ethyl, n-propyl,i-propyl, n-butyl, s-butyl or t-butyl. Yet even more preferably R¹³ ismethyl.

In another preferred embodiment, Q¹ is a diazo group. Preferably, thediazo group is according to formula (9x) as shown below, wherein R¹⁴ isselected from the group consisting of hydrogen or a carbonyl derivative.More preferably, R¹⁴ is hydrogen.

In another preferred embodiment, Q¹ is a ketone group. More preferably,the ketone group is according to formula (9y) as shown below, whereinR¹⁵ is selected from the group consisting of linear or branched C₁-C₁₂alkyl groups and C₆-C₁₂ aryl groups. Preferably, R¹⁵ is a C₁-C₆ alkylgroup, more preferably R¹⁵ is a C₁-C₄ alkyl group. Even more preferablyR¹⁵ is methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl or t-butyl.Yet even more preferably R¹⁵ is methyl.

In another preferred embodiment, Q¹ is an (O-alkyl)hydroxylamino group.More preferably, the (O-alkyl)hydroxylamino group is according toformula (9z) as shown below.

In another preferred embodiment, Q¹ is a hydrazine group. Preferably,the hydrazine group is according to formula (9za) as shown below.

In another preferred embodiment, Q¹ is a halogenated N-maleimidyl groupor a sulfonylated N-maleimidyl group. When Q¹ is a halogenated orsulfonylated N-maleimidyl group, Q¹ is preferably according to formula(9ze) as shown below, wherein R⁶ is independently selected from thegroup consisting of hydrogen F, Cl, Br, I, —SR^(18a) and —OS(O)₂R^(18b),wherein R^(18a) is an optionally substituted C₄-C₁₂ (hetero)aryl groups,preferably phenyl or pyrydyl, and R^(18b) is selected from the groupconsisting of, optionally substituted, C₁-C₁₂ alkyl groups and C₄-C₁₂(hetero)aryl groups, preferably tolyl or methyl, and with the provisothat at least one R⁶ is not hydrogen. When R⁶ is halogen (i.e. when R⁶is F, Cl, Br or I), it is preferred that R⁶ is Br. In one embodiment,the halogenated N-maleimidyl group is halogenated 2,3-diaminopropionicacid (DPR) maleimidyl, which may be connected to the remainder of thelinker-conjugate through the carboxylic acid moiety or the remainingamine moiety.

In another preferred embodiment, Q¹ is a carbonyl halide group accordingto formula (9zf) as shown below, wherein X is selected from the groupconsisting of F, Cl, Br and I. Preferably, X is Cl or Br, and mostpreferably, X is C₁.

In another preferred embodiment, Q¹ is an allenamide group according toformula (9zg).

In another preferred embodiment, Q¹ is a1,1-bis(sulfonylmethyl)methylcarbonyl group according to formula (9zh),or an elimination derivative thereof, wherein R¹⁸ is selected from thegroup consisting of, optionally substituted, C₁-C₁₂ alkyl groups andC₄-C₁₂ (hetero)aryl groups. More preferably, R₁₈ is an, optionallysubstituted, C₁-C₆ alkyl group or a C₄-C₆ (hetero)aryl group, and mostpreferably a phenyl group.

wherein k, l, X, T, U, V, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³,R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸ and R¹⁹ are as defined above.

In a preferred embodiment of the conjugation process according to theinvention as described hereinbelow, conjugation is accomplished via acycloaddition, such as a Diels-Alder reaction or a 1,3-dipolarcycloaddition, preferably the 1,3-dipolar cycloaddition. According tothis embodiment, the reactive group Q¹ (as well as F¹ on thebiomolecule) is selected from groups reactive in a cycloadditionreaction. Herein, reactive groups Q¹ and F¹ are complementary, i.e. theyare capable of reacting with each other in a cycloaddition reaction.

For a Diels-Alder reaction, one of F¹ and Q¹ is a diene and the other ofF¹ and Q¹ is a dienophile. As appreciated by the skilled person, theterm “diene” in the context of the Diels-Alder reaction refers to1,3-(hetero)dienes, and includes conjugated dienes (R₂C═CR—CR═CR₂),imines (e.g. R₂C═CR—N═CR₂ or R₂C═CR—CR═NR, R₂C═N—N═CR₂) and carbonyls(e.g. R₂C═CR—CR═O or O═CR—CR═O). Hetero-Diels-Alder reactions with N-and O-containing dienes are known in the art. Any diene known in the artto be suitable for Diels-Alder reactions may be used as reactive groupQ¹ or F¹. Preferred dienes include tetrazines as described above,1,2-quinones as described above and triazines as described above.Although any dienophile known in the art to be suitable for Diels-Alderreactions may be used as reactive groups Q¹ or F¹, the dienophile ispreferably an alkene or alkyne group as described above, most preferablyan alkyne group. For conjugation via a Diels-Alder reaction, it ispreferred that F¹ is the diene and Q¹ is the dienophile. Herein, when Q¹is a diene, F¹ is a dienophile and when Q¹ is a dienophile, F¹ is adiene. Most preferably, Q¹ is a dienophile, preferably Q¹ is orcomprises an alkynyl group, and F¹ is a diene, preferably a tetrazine,1,2-quinone or triazine group.

For a 1,3-dipolar cycloaddition, one of F¹ and Q¹ is a 1,3-dipole andthe other of F¹ and Q¹ is a dipolarophile. Any 1,3-dipole known in theart to be suitable for 1,3-dipolar cycloadditions may be used asreactive group Q¹ or F¹. Preferred 1,3-dipoles include azido groups,nitrone groups, nitrile oxide groups, nitrile imine groups and diazogroups. Although any dipolarophile known in the art to be suitable for1,3-dipolar cycloadditions may be used as reactive groups Q¹ or F¹, thedipolarophile is preferably an alkene or alkyne group, most preferablyan alkyne group. For conjugation via a 1,3-dipolar cycloaddition, it ispreferred that F¹ is the 1,3-dipole and Q¹ is the dipolarophile. Herein,when Q¹ is a 1,3-dipole, F¹ is a dipolarophile and when Q¹ is adipolarophile, F¹ is a 1,3-dipole. Most preferably, Q¹ is adipolarophile, preferably Q¹ is or comprises an alkynyl group, and F¹ isa 1,3-dipole, preferably an azido group.

Thus, in a preferred embodiment, Q¹ is selected from dipolarophiles anddienophiles. Preferably, Q¹ is an alkene or an alkyne group. In anespecially preferred embodiment, Q¹ comprises an alkyne group,preferably selected from the alkynyl group as described above, thecycloalkenyl group as described above, the (hetero)cycloalkynyl group asdescribed above and a bicyclo[6.1.0]non-4-yn-9-yl] group, morepreferably Q¹ is selected from the formulae (9j), (9n), (9o), (9p), (9q)and (9zk), as defined above and depicted below, more preferably selectedfrom the formulae (9n), (9o), (9p), (9q) and (9zk). Most preferably, Q¹is a bicyclo[6.1.0]non-4-yn-9-yl]group, preferably of formula (9q).These groups are known to be highly effective in the conjugation withazido-functionalized biomolecules as described herein, and when thesulfamide linker according to the invention is employed in suchlinker-conjugates and bioconjugates, any aggregation is beneficiallyreduced to a minimum. The sulfamide linker according to the inventionprovides a significant reduction in aggregation especially for suchhydrophobic reactive groups of Q¹, and for the conjugated bioconjugates.

As was described above, in the compound according to the invention, Q¹is capable of reacting with a reactive group F¹ that is present on abiomolecule. Complementary reactive groups F¹ for reactive group Q¹ areknown to a person skilled in the art, and are described in more detailbelow. Some representative examples of reaction between F¹ and Q¹ andtheir corresponding products are depicted in FIG. 6.

As described above, D and Q¹ are covalently attached in thelinker-conjugate according to the invention, via the linker as definedabove. Covalent attachment of D to the linker may occur for example viareaction of a functional group F² present on D with a reactive group Q²present on the linker. Suitable organic reactions for the attachment ofD to a linker are known to a person skilled in the art, as arefunctional groups F² that are complementary to a reactive group Q².Consequently, D may be attached to the linker via a connecting group Z.

The term “connecting group” herein refers to the structural element,e.g. resulting from the reaction between Q and F, connecting one part ofa compound and another part of the same compound. As will be understoodby the person skilled in the art, the nature of a connecting groupdepends on the type of organic reaction with which the connectionbetween the parts of said compound was obtained. As an example, when thecarboxyl group of R—C(O)—OH is reacted with the amino group of H₂N—R′ toform R—C(O)—N(H)—R′, R is connected to R′ via connecting group Z, and Zmay be represented by the group —C(O)—N(H)—.

Reactive group Q¹ may be attached to the linker in a similar manner.Consequently, Q¹ may be attached to the spacer-moiety via a connectinggroup Z.

Numerous reactions are known in the art for the attachment of an activesubstance to a linker, and for the attachment of a reactive group Q¹ toa linker. Consequently, a wide variety of connecting groups Z may bepresent in the first precursor. In one embodiment, the reactive group Q²is selected from the options for reactive group Q¹ and preferredembodiments thereof, as defined hereinabove, preferably as depicted inFIG. 6. Complementary functional groups F² and the thus obtainedconnecting groups Z are known to a person skilled in the art anddisclosed herein.

Preferred Linker-Conjugates

The linker-conjugate according to the invention may also be representedby the formula:(Q¹)_(y)-(Z^(w))-Sp-(Z^(x))-(D)z

wherein:

-   -   y is an integer in the range of 1 to 10, preferably in the range        1-5, more preferably y=1;    -   z is an integer in the range of 1 to 10, preferably in the range        1-5;    -   Q¹ is a reactive group as defined hereinabove;    -   D is a target molecule as defined hereinabove;    -   Sp is a spacer moiety, wherein a spacer moiety is defined as a        moiety that spaces (i.e. provides a certain distance between)        and covalently links reactive group Q¹ and target molecule D,        wherein said spacer moiety comprises the linker according to the        invention as defined above;    -   Z^(w) is a connecting group connecting reactive group Q¹ to said        spacer moiety; and    -   Z^(x) is a connecting group connecting target molecule D to said        spacer moiety.

Preferred embodiments for y and z are as defined above for(Q¹)_(y)-Sp-(D)_(z). Preferred embodiments for D and Q¹ are as definedabove. It is further preferred that the compound is according to theformula Q¹-(Z^(w))-Sp-(Z^(x))-(D)₄, Q¹-(Z^(w))-Sp-(Z^(x))-(D)₃,Q¹-(Z^(w))-Sp-(Z^(x))-(D)₂ or Q¹-(Z^(w))-Sp-(Z^(x))-D, more preferablyQ¹-(Z^(w))-Sp-(Z^(x))-(D)₂ or Q¹-(Z^(w))-Sp-(Z^(x))-D and mostpreferably Q¹-(Z^(w))-Sp-(Z^(x))-D, wherein Z^(w) and Z^(x) are asdefined above.

Preferably, Z^(w) and Z^(x) are independently selected from the groupconsisting of —O—, —S—, —NR²—, —N═N—, —C(O)—, —C(O)—NR²—, —O—C(O)—,—O—C(O)—O—, —O—C(O)—NR², —NR²—C(O)—, —NR²—C(O)—O—, —NR²—C(O)—NR²—,—S—C(O)—, —S—C(O)—O—, —S—C(O)—NR²—, —S(O)—, —S(O)₂—, —O—S(O)₂—,—O—S(O)₂—O—, —O—S(O)₂—NR²—, —O—S(O)—, —O—S(O)—O—, —O—S(O)—NR²—,—O—NR²—C(O)—, —O—NR²—C(O)—O—, —O—NR²—C(O)—NR²—, —NR²—O—C(O)—,—NR²—O—C(O)—O—, —NR²—O—C(O)—NR²—, —O—NR²—C(S)—, —O—NR²—C(S)—O—,—O—NR²—C(S)—NR²—, —NR²—O—C(S)—, —NR²—O—C(S)—O—, —NR²—O—C(S)—NR²—,—O—C(S)—, —O—C(S)—O—, —O—C(S)—NR²—, —NR²—C(S)—, —NR²—C(S)—O—,—NR²—C(S)—NR²—, —S—S(O)₂—, —S—S(O)₂—O—, —S—S(O)₂—NR²—, —NR²—O—S(O)—,—NR²—O—S(O)—O—, —NR²—O—S(O)—NR²—, —NR²—O—S(O)₂—, —NR²—O—S(O)₂—O—,—NR²—O—S(O)₂—NR²—, —O—NR²—S(O)—, —O—NR²—S(O)—O—, —O—NR²—S(O)—NR²—,—O—NR²—S(O)₂—O—, —O—NR²—S(O)₂—NR²—, —O—NR²—S(O)₂—, —O—P(O)(R²)₂—,—S—P(O)(R²)₂—, —NR²—P(O)(R²)₂— and combinations of two or more thereof,wherein R² is independently selected from the group consisting ofhydrogen, C₁-C₂₄ alkyl groups, C₂-C₂₄ alkenyl groups, C₂-C₂₄ alkynylgroups and C₃-C₂₄ cycloalkyl groups, the alkyl groups, alkenyl groups,alkynyl groups and cycloalkyl groups being optionally substituted.

The linker-conjugate according to the invention may also be representedby formula (4):

Herein:

-   -   BM is a branching moiety as defined above;    -   E is a capping group as defined above;    -   SG is a sulfamide group according to formula (1), wherein a and        R¹ are as defined above;    -   D is a target molecule as defined above;    -   Q¹ is a reactive group as defined above;    -   b is independently 0 or 1;    -   c is 0 or 1;    -   d is 0 or 1;    -   e is 0 or 1;    -   f is an integer in the range of 1 to 10;    -   g is 0 or 1;    -   i is 0 or 1;    -   k is 0 or 1;    -   l is 0 or 1;    -   Sp¹ is a spacer moiety;    -   Sp² is a spacer moiety;    -   Sp³ is a spacer moiety;    -   Sp⁴ is a spacer moiety;    -   Sp⁵ is a spacer moiety;    -   Sp⁶ is a spacer moiety;    -   Z¹ is a connecting group that connects Q¹ or Sp³ to Sp² or BM;        and    -   Z² is a connecting group that connects D or Sp⁴ to Sp¹ or BM.

Definitions and preferred embodiments for each of BM, E, SG, b, c, d, e,f, g, i, k, l, Sp¹, Sp², Sp³, Sp⁴, Sp⁵, Sp⁶, Z¹ and Z² are given abovefor the linker, and also apply to the linker-conjugate according tostructure (4). Definitions and preferred embodiments for each of Q¹ andBM are given above, and also apply to the linker-conjugate according tostructure (4).

Especially preferred linker-conjugates are represented by the structuresby any of the structures (34)-(39):

In structures (34)-(39), n and m are individually 0-20, preferably 0-5,most preferably 0 or 1, and Q¹, D and E are as defined above. In oneembodiment, the linker-conjugate is represented by structure (34),wherein n=0 (corresponding to compound 32 as prepared in Example 9).

The invention further concerns the use of a linker-conjugate accordingto the invention for the preparation of a bioconjugate according to theinvention. Such a process for the preparation is typically referred toas “bioconjugation” or a “bioconjugation reaction”, and is furtherdefined below.

Linker-Construct

In the context of the present invention, “linker-construct” refers to acompound wherein a reactive group Q¹ is covalently connected to areactive group Q² via a linker according to the invention, comprising abranching moiety BM and a sulfamide group SG. In other words, thelinker-construct is a compound wherein a reactive group Q² is covalentlyconnected to a reactive group Q¹, via a linker. In a second aspect, theinvention concerns linker-constructs as defined herein. Thelinker-construct according to the invention comprises the linkeraccording to the invention as defined above, preferably the linkeraccording to structure (2).

A linker-construct comprises a reactive group Q¹ capable of reactingwith a reactive group F¹ present on a biomolecule, and a reactive groupQ² capable of reacting with a reactive group F² present on a targetmolecule. Q¹ and Q² may be the same, or different. A linker-constructmay comprise more than one reactive group Q¹ and/or more than onereactive group Q². When more than one reactive groups Q¹ are present thegroups Q¹ may be the same or different, and when more than one reactivegroups Q² are present the groups Q² may be the same or different.

The linker-construct according to the invention may be denoted as(Q¹)_(y)-Sp-(Q²)_(z), wherein y is an integer in the range of 1 to 10and z is an integer in the range of 1 to 10.

The invention thus also relates to a linker-construct according to theformula (Q¹)_(y)-Sp-(Q²)_(z), wherein:

-   -   y is an integer in the range of 1 to 10, preferably in the range        1-5, more preferably y=1;    -   z is an integer in the range of 1 to 10, preferably in the range        1-5;    -   Q¹ is a reactive group as defined hereinabove;    -   Q² is a reactive group capable of reacting with a functional        group F² present on a target molecule;    -   Sp is a spacer moiety, wherein a spacer moiety is defined as a        moiety that spaces (i.e. provides a certain distance between)        and covalently links reactive group Q¹ and target molecule Q²,        wherein said spacer moiety comprises the linker according to the        invention as defined above.

Preferably, y is 1, 2, 3 or 4, more preferably y is 1 or 2 and mostpreferably, y is 1. Preferably, z is 1, 2, 3, 4, 5 or 6, more preferablyz is 1, 2, 3 or 4, even more preferably z is 1, 2 or 3, yet even morepreferably z is 1 or 2 and most preferably z is 1. More preferably, y is1 or 2, preferably 1, and z is 1, 2, 3 or 4, yet even more preferably yis 1 or 2, preferably 1, and z is 1, 2 or 3, yet even more preferably yis 1 or 2, preferably 1, and z is 1 or 2, and most preferably y is 1 andz is 1. In a preferred embodiment, the linker-construct is according tothe formula Q¹-Sp-(Q²)₄, Q¹-Sp-(Q²)₃, Q¹-Sp-(Q²)₂ or Q¹-Sp-Q².

More particular, the linker-construct according to the invention may berepresented by the formula: (Q¹)_(y)-(Z^(w))—Sp-(Z)_(Q²)_(z), wherein:

-   -   y is an integer in the range of 1 to 10, preferably in the range        1-5, more preferably y=1;    -   z is an integer in the range of 1 to 10, preferably in the range        1-5;    -   Q¹ is a reactive group as defined hereinabove;    -   Q² is a reactive group capable of reacting with a functional        group F² present on a target molecule;    -   Sp is a spacer moiety, wherein a spacer moiety is defined as a        moiety that spaces (i.e. provides a certain distance between)        and covalently links reactive group Q¹ and reactive group Q²,        wherein said spacer moiety comprises the linker according to the        invention as defined above;    -   Z is a connecting group connecting reactive group Q¹ to said        spacer moiety; and    -   Z^(x) is a connecting group connecting reactive group Q² to said        spacer moiety.

Preferred embodiments for y and z are as defined above for(Q¹)_(y)-Sp-(Q²)_(z). It is further preferred that the compound isaccording to the formula Q¹-(Z^(w))-Sp-(Z^(x))-(Q²)₄,Q¹-(Z^(w))-Sp-(Z^(x))-(Q²)₃, Q¹-(Z^(w))-Sp-(Z^(x))-(Q²)₂ orQ¹-(Z^(w))-Sp-(Z^(x))-Q², more preferably Q¹-(Z^(w))-Sp-(Z^(x))-(Q²)₂ orQ¹-(Z^(w))-Sp-(Z^(x))-Q² and most preferably Q¹-(Z^(w))-Sp-(Z^(x))-Q²,wherein Z^(w) and Z^(x) are as defined above.

In the linker compound according to the invention, Z^(w) and Z^(x) arepreferably independently selected from the group consisting of —O—, —S—,—NR²—, —N═N—, —C(O)—, —C(O)—NR²—, —O—C(O)—, —O—C(O)—O—, —O—C(O)—NR²,—NR²—C(O)—, —NR²—C(O)—O—, —NR²—C(O)—NR²—, —S—C(O)—, —S—C(O)—O—,—S—C(O)—NR²—, —S(O)—, —S(O)₂—, —O—S(O)₂—, —O—S(O)₂—O—, —O—S(O)₂—NR²—,—O—S(O)—, —O—S(O)—O—, —O—S(O)—NR²—, —O—NR²—C(O)—, —O—NR²⁻C(O)—O—,—O—NR²—C(O)—NR²—, —NR²—O—C(O)—, —NR²—O—C(O)—O—, —NR²—O—C(O)—NR²—,—O—NR²—C(S)—, —O—NR²—C(S)—O—, —O—NR²—C(S)—NR²—, —NR²—O—C(S)—,—NR²—O—C(S)—O—, —NR²—O—C(S)—NR²—, —O—C(S)—, —O—C(S)—O—, —O—C(S)—NR²—,—NR²—C(S)—, —NR²—C(S)—O—, —NR²—C(S)—NR²—, —S—S(O)₂—, —S—S(O)₂—O—,—S—S(O)₂—NR²—, —NR²—O—S(O)—, —NR²—O—S(O)—O—, —NR²—O—S(O)—NR²—,—NR²—O—S(O)₂—, —NR²—O—S(O)₂—O—, —NR²—O—S(O)₂—NR²—, —O—NR²—S(O)—,—O—NR²—S(O)—O—, —O—NR²—S(O)—NR²—, —O—NR²—S(O)₂—O—, —O—NR²—S(O)₂—NR²—,—O—NR²—S(O)₂—, —O—P(O)(R²)₂—, —S—P(O)(R²)₂—, —NR²—P(O)(R²)₂— andcombinations of two or more thereof, wherein R² is independentlyselected from the group consisting of hydrogen, C₁-C₂₄ alkyl groups,C₂-C₂₄ alkenyl groups, C₂-C₂₄ alkynyl groups and C₃-C₂₄ cycloalkylgroups, the alkyl groups, alkenyl groups, alkynyl groups and cycloalkylgroups being optionally substituted.

Preferred embodiments for Q¹ are as defined above for thelinker-conjugate. In the linker-construct according to the invention, Q²is a reactive group capable of reacting with a functional group F²present on a target molecule. Reactive groups Q² capable of reactingwith such a functional group F² are known to a person skilled in theart. In a preferred embodiment, Q² is a reactive group selected from thegroup consisting of, optionally substituted, N-maleimidyl groups,halogenated N-alkylamido groups, sulfonyloxy N-alkylamido groups, estergroups, carbonate groups, sulfonyl halide groups, thiol groups orderivatives thereof, alkenyl groups, alkynyl groups,(hetero)cycloalkynyl groups, bicyclo[6.1.0]non-4-yn-9-yl] groups,cycloalkenyl groups, tetrazinyl groups, azido groups, phosphine groups,nitrile oxide groups, nitrone groups, nitrile imine groups, diazogroups, ketone groups, (O-alkyl)hydroxylamino groups, hydrazine groups,halogenated N-maleimidyl groups, 1,1-bis(sulfonylmethyl)methylcarbonylgroups or elimination derivatives thereof, carbonyl halide groups andallenamide groups.

In a further preferred embodiment, Q² is according to formula (9a),(9b), (9c), (9d), (9e), (9f), (9g), (9h), (9i), (9j), (9k), (9l), (9m),(9n), (9o), (9p), (9q), (9r), (9s), (9t), (9u), (9v), (9w), (9x), (9y),(9z), (9za), (9zb), (9zc), (9zd), (9ze), (9zf), (9zg), (9zh), (9zi),(9zj), (9zk), (9zl), (9zm) or (9zn), wherein (9a), (9b), (9c), (9d),(9e), (9f), (9g), (9h), (9i), (9j), (9k), (9l), (9m), (9n), (9o), (9p),(9q), (9r), (9s), (9t), (9u), (9v), (9w), (9x), (9y), (9z), (9za),(9zb), (9zc), (9zd), (9ze), (9zf), (9zg), (9zh), (9zi), (9zj), (9zk),(9zl), (9zm), (9zn) and preferred embodiments thereof, are as definedabove. In this embodiment it is further preferred that Q² is accordingto formula (9a), (9b), (9c), (9f), (9j), (9n), (9o), (9p), (9q), (9s),(9t), (9zh), (9r), (9zl), (9zm) or (9zn), more preferably according toformula (9a), (9n), (9o), (9p), (9q), (9t), (9zh) or (9s), and even morepreferably Q² is according to formula (9a), (9p), (9q), (9n), (9t),(9zh), or (9o), and preferred embodiments thereof, as defined above.Most preferably, Q² is according to formula (9a), (9p), (9q), (9n),(9t), (9zh) or (9o), and preferred embodiments thereof, as definedabove.

In a specific embodiment of the linker-construct according to theinvention, Q¹ is according to formula (9a), (9b), (9c), (9d), (9e),(9f), (9g), (9h), (9i), (9j), (9k), (9l), (9m), (9n), (9o), (9p), (9q),(9r), (9s), (9t), (9u), (9v), (9w), (9x), (9y), (9z), (9za), (9zb),(9zc), (9zd), (9ze), (9zf), (9zg), (9zh), (9zi), (9zj), (9zk), (9zl),(9zm) or (9zn), wherein (9a), (9b), (9c), (9d), (9e), (9f), (9g), (9h),(9i), (9j), (9k), (9l), (9m), (9n), (9o), (9p), (9q), (9r), (9s), (9t),(9u), (9v), (9w), (9x), (9y), (9z), (9za), (9zb), (9zc), (9zd), (9ze),(9zf), (9zg), (9zh), (9zi), (9zj), (9zk), (9zl), (9zm), (9zn) andpreferred embodiments thereof, are as defined above; and spacer moietySp is selected from the group consisting of linear or branched C₁-C₂₀alkylene groups, the alkylene groups being optionally substituted andoptionally interrupted by one or more heteroatoms selected from thegroup consisting of O, S and NR³, wherein R³ is independently selectedfrom the group consisting of hydrogen and C₁-C₄ alkyl groups; whereinthe spacer moiety Sp is interrupted by one or more branching moietiesBM, connected to a sulfamide group SG and capping moiety E, optionallyvie one or two spacers, as defined above.

In a further preferred embodiment, Q¹ is according to formula (9a),(9b), (9c), (9f), (9j), (9n), (9o), (9p), (9q), (9s), (9t), (9zh),(9zk), (9r), (9zl), (9zm) or (9zn). In an even further preferredembodiment, Q¹ is according to formula (9a), (9n), (9o), (9p), (9q),(9t), (9zh) or (9s), and in a particularly preferred embodiment, Q¹ isaccording to formula (9a), (9p), (9q), (9n), (9t), (9zh), (9zk) or (9o),and preferred embodiments thereof, as defined above. Most preferably Q¹is according to formula (9a), (9p), (9q), (9n), (9t), (9zh) or (9o), andpreferred embodiments thereof, as defined above.

The invention further concerns the use of a linker-construct accordingto the invention for the preparation of a linker-conjugate according tothe invention. The process for preparing the linker-conjugate accordingto the invention is further defined below.

The invention further relates to the use of a linker-construct accordingto the invention in a bioconjugation process. The linker-constructaccording to the invention, and preferred embodiments therefore, aredescribed in detail above.

Process for the Preparation of a Linker-Conjugate

The present invention also relates to a process for the preparation of alinker-conjugate according to the invention. In particular, theinvention relates to a process for the preparation of a linker-conjugateaccording to the invention, the process comprising the step of reactinga functional group Q² of a linker-construct with a functional group F²of a target molecule, wherein said linker-construct is as defined above.The linker-construct and preferred embodiments thereof, includingpreferred embodiments of Q¹, Q² and target molecule D, are described indetail above. In a preferred embodiment of the process for thepreparation of a linker-conjugate, the linker-construct is according to(Q¹)_(y)-Sp-(Q²)_(z) as defined above. In a further preferred embodimentof the process for the preparation of a linker-conjugate, thelinker-construct is according to (Q¹)_(y)-(Z^(w))—Sp-(Z^(x))-(Q²)_(z) asdefined above.

Bioconjugate

In a third aspect, the invention concerns bioconjugates as definedherein. In the context of the present invention, “bioconjugate” refersto a compound wherein a biomolecule is covalently connected to a targetmolecule via a linker according to the invention, comprising a branchingmoiety BM and a sulfamide group SG. In other words, the bioconjugate isa compound wherein a biomolecule is covalently connected to a targetmolecule, via a linker. A bioconjugate comprises one or morebiomolecules and/or one or more target molecules. The bioconjugateaccording to the invention comprises the linker according to theinvention as defined above, preferably the linker according to structure(2).

The bioconjugate according to the invention is typically prepared by theprocess for preparation of a bioconjugate according to the invention,wherein the linker-conjugate comprising reactive group Q¹ is conjugatedto a biomolecule comprising reactive group F¹. In this conjugationreaction, reactive groups Q¹ and F¹ react with each other to form aconnecting group Z³, which joins the linker-conjugate with thebiomolecule. All preferred embodiments described herein for thelinker-conjugate and the linker thus equally apply to the bioconjugateaccording to the invention, except for all said for Q¹ and F, as thebioconjugate according to the invention contains the reaction product ofQ¹ and F¹ as defined herein.

The bioconjugate according to the invention may comprise more than onetarget molecule. Similarly, the bioconjugate may comprise more than onebiomolecule. Biomolecule B and target molecule D, and preferredembodiments thereof, are described in more detail above. Preferredembodiments for D in the bioconjugate according to the inventioncorrespond to preferred embodiments of D in the linker-conjugateaccording to the invention as were described in more detail above.Preferred embodiments for the linker according to the invention asdescribed above also apply to the linker comprised in the bioconjugateaccording to the invention. In the bioconjugate according to theinvention, biomolecule B is preferably selected from the groupconsisting of proteins (including glycoproteins and antibodies),polypeptides, peptides, glycans, lipids, nucleic acids,oligonucleotides, polysaccharides, oligosaccharides, enzymes, hormones,amino acids and monosaccharides. More preferably, biomolecule B isselected from the group consisting of proteins (including glycoproteinsand antibodies), polypeptides, peptides, glycans, nucleic acids,oligonucleotides, polysaccharides, oligosaccharides and enzymes. Mostpreferably, biomolecule B is selected from the group consisting ofproteins, including glycoproteins and antibodies, polypeptides, peptidesand glycans.

The bioconjugate according to the invention may also be defined as abioconjugate wherein a biomolecule is conjugated to a target moleculevia a spacer-moiety, wherein the spacer-moiety comprises the linkeraccording to the invention. The bioconjugate according to the inventionmay also be denoted as (B)_(y)—Sp-(D)_(z), wherein y is an integer inthe range of 1 to 10 and z is an integer in the range of 1 to 10. Theinvention thus also relates to a bioconjugate according to the formula:(B)_(y)—Sp-(D)_(z), wherein:

-   -   y is an integer in the range of 1 to 10, preferably in the range        1-5, more preferably y=1;    -   z is an integer in the range of 1 to 10, preferably in the range        1-5;    -   B is a biomolecule as defined herein;    -   D is a target molecule as defined herein; and    -   Sp is a spacer moiety, wherein a spacer moiety is defined as a        moiety that spaces (i.e. provides a certain distance between)        and covalently links biomolecule B and target molecule D,        wherein said spacer moiety comprises the linker according to the        invention as defined above.

Preferably, y is 1, 2, 3 or 4, more preferably y is 1 or 2 and mostpreferably, y is 1. Preferably, z is 1, 2, 3, 4, 5 or 6, more preferablyz is 1, 2, 3 or 4, even more preferably z is 1, 2 or 3, yet even morepreferably z is 1 or 2 and most preferably z is 1. More preferably, y is1 or 2, preferably 1, and z is 1, 2, 3 or 4, yet even more preferably yis 1 or 2, preferably 1, and z is 1, 2 or 3, yet even more preferably yis 1 or 2, preferably 1, and z is 1 or 2, and most preferably y is 1 andz is 1. In a preferred embodiment, the bioconjugate is according to theformula B-Sp-(D)₄, B-Sp-(D)₃, B-Sp-(D)₂ or B-Sp-D.

In a preferred embodiment, the bioconjugate according to the inventionis represented by formula (5):

Herein, b, c, d, e, f, g, i, D, BM, SG, E, Sp¹, Sp², Sp³, Sp⁴, Sp⁵, Sp⁶,Z¹ and Z², and preferred embodiments thereof, are as defined above forthe linker according to the invention or the linker-conjugate of theinvention, and

-   -   h is 0 or 1;    -   j is 0 or 1;    -   Z³ is a connecting group that connects B or Sp⁷ to Sp³, Z¹, Sp²        or BM;    -   Sp⁷ is a spacer moiety; and    -   B is a biomolecule as defined herein.

Preferably, h is 1. Preferably, j is 0. Preferred embodiments ofbiomolecule B are as defined above. Preferred embodiment for Sp⁷ areaccording to the preferred embodiments of any one Sp¹-Sp⁶ as definedabove. Z³ is a connecting group. As described above, the term“connecting group” herein refers to the structural element connectingone part of a compound and another part of the same compound. Typically,a bioconjugate is prepared via reaction of a reactive group Q¹ presentin a linker-conjugate with a functional group F¹ present in abiomolecule. As will be understood by the person skilled in the art, thenature of connecting group Z³ depends on the type of organic reactionthat was used to establish the connection between a biomolecule and alinker-conjugate. In other words, the nature of Z³ depends on the natureof reactive group Q¹ of the linker-conjugate and the nature offunctional group F¹ in the biomolecule. Since there is a large number ofdifferent chemical reactions available for establishing the connectionbetween a biomolecule and a linker-conjugate, consequently there is alarge number of possibilities for Z³.

Several examples of suitable combinations of F¹ and Q¹, and ofconnecting group Z³ that will be present in a bioconjugate when alinker-conjugate comprising Q¹ is conjugated to a biomolecule comprisinga complementary functional group F, are shown in FIG. 6.

When F¹ is for example a thiol group, complementary groups Q¹ includeN-maleimidyl groups and alkenyl groups, and the corresponding connectinggroups Z³ are as shown in FIG. 6. When F¹ is a thiol group,complementary groups Q¹ also include allenamide groups.

When F¹ is for example an amino group, complementary groups Q¹ includeketone groups and activated ester groups, and the correspondingconnecting groups Z³ are as shown in FIG. 6.

When F¹ is for example a ketone group, complementary groups Q¹ include(O-alkyl)hydroxylamino groups and hydrazine groups, and thecorresponding connecting groups Z³ are as shown in FIG. 6.

When F¹ is for example an alkynyl group, complementary groups Q¹ includeazido groups, and the corresponding connecting group Z³ is as shown inFIG. 6.

When F¹ is for example an azido group, complementary groups Q¹ includealkynyl groups, and the corresponding connecting group Z³ is as shown inFIG. 6.

When F¹ is for example a cyclopropenyl group, a trans-cyclooctene groupor a cyclooctyne group, complementary groups Q¹ include tetrazinylgroups, and the corresponding connecting group Z³ is as shown in FIG. 6.In these particular cases, Z³ is only an intermediate structure and willexpel N₂, thereby generating a dihydropyridazine (from the reaction withalkene) or pyridazine (from the reaction with alkyne).

Additional suitable combinations of F¹ and Q¹, and the nature ofresulting connecting group Z³ are known to a person skilled in the art,and are e.g. described in G. T. Hermanson, “Bioconjugate Techniques”,Elsevier, 3^(rd) Ed. 2013 (ISBN: 978-0-12-382239-0), in particular inChapter 3, pages 229-258, incorporated by reference. A list ofcomplementary reactive groups suitable for bioconjugation processes isdisclosed in Table 3.1, pages 230-232 of Chapter 3 of G. T. Hermanson,“Bioconjugate Techniques”, Elsevier, 3^(rd) Ed. 2013 (ISBN:978-0-12-382239-0), and the content of this Table is expresslyincorporated by reference herein.

In the bioconjugate according to (5), it is preferred that at least oneof Z³, Sp³, Z¹ and Sp² is present, i.e. at least one of h, g, d and c isnot 0. It is also preferred that at least one of Sp¹, Z² and Sp⁴ ispresent, i.e. that at least one of b, e and i is not 0. More preferably,at least one of Z³, Sp³, Z¹ and Sp² is present and at least one of Sp,Z² and Sp⁴ is present, i.e. it is preferred that at least one of b, eand i is not 0 and at least one of h, g, d and c is not 0.

Process for the Preparation of a Bioconjugate

In a fourth aspect, the present invention also relates to a process forthe preparation of a bioconjugate, the process comprising the step ofreacting a reactive group Q¹ of a linker-conjugate according to theinvention with a functional group F¹ of a biomolecule. Thelinker-conjugate according to the invention, and preferred embodimentsthereof, are described in more detail above.

FIG. 1 shows the general concept of conjugation of biomolecules: abiomolecule of interest (BOI) comprising one or more functional groupsF¹ is incubated with (excess of) a target molecule D (also referred toas molecule of interest or MOI) covalently attached to a reactive groupQ¹ via a specific linker. In the process of bioconjugation, a chemicalreaction between F¹ and Q¹ takes place, thereby forming a bioconjugatecomprising a covalent bond between the BOI and the MOI. The BOI may e.g.be a peptide/protein, a glycan or a nucleic acid. In the processaccording to the invention, the linker is a sulfamide linker.

The present invention thus relates to a process for the preparation of abioconjugate, the process comprising the step of reacting a reactivegroup Q¹ of a linker-conjugate with a functional group F¹ of abiomolecule, wherein the linker-conjugate is the linker-conjugateaccording to the invention as defined above and comprises the linkeraccording to the invention as defined above. The present process occursunder condition such that reactive group Q¹ of the linker-conjugate isreacted with the functional group F¹ of the biomolecule to covalentlylink the biomolecule to the linker-conjugate. In the process accordingto the invention, Q¹ reacts with F, forming a covalent connectionbetween the biomolecule and the linker-moiety. Complementary reactivegroups Q¹ and functional groups F¹ are described in more detail aboveand below.

In a preferred embodiment, the process according to this aspect of theinvention concerns a process for the preparation of a bioconjugate via acycloaddition or a Michael reaction. A preferred Michael reaction is thethiol-maleimide addition, most preferably wherein Q¹ is maleimide and F¹is a thiol group. Preferred cycloadditions are a (4+2)-cycloaddition(e.g. a Diels-Alder reaction) or a (3+2)-cycloaddition (e.g. a1,3-dipolar cycloaddition). Preferably, the conjugation is theDiels-Alder reaction or the 1,3-dipolar cycloaddition. The preferredDiels-Alder reaction is the inverse-electron demand Diels-Aldercycloaddition. In another preferred embodiment, the 1,3-dipolarcycloaddition is used, more preferably the alkyne-azide cycloaddition,and most preferably wherein Q¹ is or comprises an alkyne group and F¹ isan azido group. Cycloadditions, such as Diels-Alder reactions and1,3-dipolar cycloadditions are known in the art, and the skilled personknowns how to perform them. In a further preferred embodiment, theinvention concerns a process for the preparation of a bioconjugate,wherein the target molecule is hydrophobic (i.e. poorly soluble inwater), most preferably wherein the target molecule has a watersolubility of at most 0.1% (w/w) in water (20° C. and 100 kPa). In anespecially preferred embodiment, the invention concerns a process forthe preparation of a bioconjugate via cycloaddition, preferably a1,3-dipolar cycloaddition, more preferably the alkyne-azidecycloaddition, and most preferably wherein Q¹ is or comprises an alkynegroup and F¹ is an azido group, and wherein the target molecule ishydrophobic, most preferably wherein the target molecule has a watersolubility of at most 0.1% (w/w) in water (20° C. and 100 kPa).

Biomolecules are described in more detail above. Preferably, in theprocess according to the invention the biomolecule is selected from thegroup consisting of proteins (including glycoproteins and antibodies),polypeptides, peptides, glycans, lipids, nucleic acids,oligonucleotides, polysaccharides, oligosaccharides, enzymes, hormones,amino acids and monosaccharides. More preferably, biomolecule B isselected from the group consisting of proteins (including glycoproteinsand antibodies), polypeptides, peptides, glycans, nucleic acids,oligonucleotides, polysaccharides, oligosaccharides and enzymes. Mostpreferably, biomolecule B is selected from the group consisting ofproteins, including glycoproteins and antibodies, polypeptides, peptidesand glycans.

Target molecules are described in more detail above. In a preferredembodiment of the process according to the invention, the targetmolecule is selected from the group consisting of an active substance, areporter molecule, a polymer, a solid surface, a hydrogel, ananoparticle, a microparticle and a biomolecule. Active substances,reporter molecules, polymers, solid surfaces, hydrogels, nanoparticlesand microparticles are described in detail above, as are their preferredembodiments. In view of the significantly improved water solubility ofthe linker-conjugate when the sulfamide linker according to theinvention is employed, a preferred embodiment of the process for thepreparation of a bioconjugate employs a hydrophobic target molecule. Thehydrophobic target molecule in its unconjugated form typically has awater solubility of at most 1% (w/w), preferably at most 0.5% (w/w),most preferably at most 0.1% (w/w), determined at 20° C. and 100 kPa.

Reactive group Q¹ are described in more detail above. In the processaccording to the invention, it is preferred that reactive group Q¹ isselected from the group consisting of, optionally substituted,N-maleimidyl groups, halogenated N-alkylamido groups, sulfonyloxyN-alkylamido groups, ester groups, carbonate groups, sulfonyl halidegroups, thiol groups or derivatives thereof, alkenyl groups, alkynylgroups, (hetero)cycloalkynyl groups, bicyclo[6.1.0]non-4-yn-9-yl]groups, cycloalkenyl groups, tetrazinyl groups, azido groups, phosphinegroups, nitrile oxide groups, nitrone groups, nitrile imine groups,diazo groups, ketone groups, (O-alkyl)hydroxylamino groups, hydrazinegroups, halogenated N-maleimidyl groups,1,1-bis(sulfonylmethyl)methylcarbonyl groups or elimination derivativesthereof, carbonyl halide groups and allenamide groups.

In a further preferred embodiment, Q¹ is according to formula (9a),(9b), (9c), (9d), (9e), (9f), (9g), (9h), (9i), (9j), (9k), (9l), (9m),(9n), (9o), (9p), (9q), (9r), (9s), (9t), (9u), (9v), (9w), (9x), (9y),(9z), (9za), (9zb), (9zc), (9zd), (9ze), (9zf), (9zg), (9zh), (9zi),(9zj), (9zk), (9zl), (9zm) or (9zn), wherein (9a), (9b), (9c), (9d),(9e), (9f), (9g), (9h), (9i), (9j), (9k), (9l), (9m), (9n), (9o), (9p),(9q), (9r), (9s), (9t), (9u), (9v), (9w), (9x), (9y), (9z), (9za),(9zb), (9zc), (9zd), (9ze), (9zf), (9zg), (9zh), (9zi), (9zj), (9zk),(9zl), (9zm), (9zn) and preferred embodiments thereof, are as definedabove for the linker-conjugate according to the invention. Morepreferably, Q¹ is according to formula (9a), (9b), (9c), (9f), (9j),(9n), (9o), (9p), (9q), (9s), (9t), (9zh), (9r), (9zl), (9zm) or (9zn).Even more preferably, Q¹ is according to formula (9a), (9n), (9o), (9p),(9q), (9t), (9zh) or (9s), and most preferably, Q¹ is according toformula (9a), (9p), (9q), (9n), (9t), (9zh) or (9o), and preferredembodiments thereof, as defined above.

In an especially preferred embodiment, Q¹ comprises an alkyne group,preferably selected from the alkynyl group as described above, thecycloalkenyl group as described above, the (hetero)cycloalkynyl group asdescribed above and a bicyclo[6.1.0]non-4-yn-9-yl] group, morepreferably Q¹ is selected from the formulae (9j), (9n), (9o), (9p), (9q)and (9zk), as defined above. Most preferably, Q¹ is abicyclo[6.1.0]non-4-yn-9-yl] group, preferably of formula (9q).

In a further preferred embodiment of the process according to theinvention, the linker-conjugate is according to formula (4), as definedabove. Definitions and preferred embodiments for each of Q¹, BM, E, SG,D b, c, d, e, f, g, i, k, l, Sp¹, Sp², Sp³, Sp⁴, Sp⁵, Sp⁶, Z¹ and Z² aregiven above for the linker and the linker-conjugate, and also apply tothe linker-conjugate used in the process according to the presentaspect.

Sp¹, Sp², Sp³, Sp⁴, Sp⁵ and Sp⁶ are, independently, spacer moieties, inother words, Sp¹, Sp², Sp³, Sp⁴, Sp⁵ and Sp⁶ may differ from each other.Sp¹, Sp², Sp³, Sp⁴, Sp⁵ and Sp⁶ may be present or absent (b, c, g, i, kand l are, independently, 0 or 1). However, it is preferred that atleast one of Sp¹, Sp², Sp³, Sp⁴, Sp⁵ and Sp⁶ is present, i.e. it ispreferred that at least one of b, c, g, i, k and l is not 0. Preferredembodiment for each of the spacers Sp¹, Sp², Sp³, Sp⁴, Sp⁵ and Sp⁶ aredescribed above for the linker according to the invention.

In a partially preferred process according to the invention, Sp¹, Sp²,Sp⁴, Sp⁵ and Sp⁶, if present, are independently selected from the groupconsisting of linear or branched C₁-C₂₀ alkylene groups, the alkylenegroups being optionally substituted and optionally interrupted by one ormore heteroatoms selected from the group consisting of O, S and NR³,wherein R³ is independently selected from the group consisting ofhydrogen and C₁-C₄ alkyl groups, and wherein Q¹ is according to formula(9a), (9p), (9q), (9n), (9t), (9zh) or (9o):

wherein:

-   -   R¹⁰ is a (thio)ester group; and    -   R¹⁸ is selected from the group consisting of, optionally        substituted, C₁-C₁₂ alkyl groups and C₄-C₁₂ (hetero)aryl groups.

As described above, in the process for the preparation of abioconjugate, a reactive group Q¹ that is present in a linker-conjugateis reacted with a functional group F¹ that is present in a biomolecule.In the process according to the invention, more than one functionalgroup may be present in the biomolecule. When two or more functionalgroups are present, said groups may be the same or different. Similarly,more than one reactive group may be present in the linker-conjugate.When two or more reactive groups are present, said groups may be thesame or different. In a preferred embodiment of the process according tothe invention, the linker-conjugate comprises one reactive group Q¹, andone or more target molecules D which may be the same or different. Thelinker-conjugate comprises for example 1, 2, 3, 4, 5 or 6, preferably 1,2, 3 or 4, more preferably 1, 2 or 3, even more preferably 1 or 2 targetmolecule D. In a particularly preferred embodiment the linker-conjugatecomprises 1 target molecule D. In another particularly preferredembodiment, the linker-conjugate comprises 2 target molecules D, whichmay be the same or different. In another preferred embodiment, thebiomolecule comprises two or more functional groups F, which may be thesame or different, and two or more functional groups react with acomplementary reactive group Q of a linker-conjugate. For example abiomolecule comprising two functional groups F, i.e. F¹ and F², mayreact with two linker-conjugates comprising a functional group Q¹, whichmay be the same or different, to form a bioconjugate. Examples of afunctional group F¹ in a biomolecule comprise an amino group, a thiolgroup, a carboxylic acid, an alcohol group, a carbonyl group, aphosphate group, or an aromatic group. The functional group in thebiomolecule may be naturally present or may be placed in the biomoleculeby a specific technique, for example a (bio)chemical or a genetictechnique. The functional group that is placed in the biomolecule may bea functional group that is naturally present in nature, or may be afunctional group that is prepared by chemical synthesis, for example anazide, a terminal alkyne, a cyclopropene moiety or a phosphine moiety.In view of the preferred mode of conjugation by cycloaddition, it ispreferred that F¹ is group capable of reacting in a cycloaddition, suchas a diene, a dienophile, a 1,3-dipole or a dipolarophile, preferably F¹is selected from a 1,3-dipole (typically an azido group, nitrone group,nitrile oxide group, nitrile imine group or diazo group) or adipolarophile (typically an alkenyl or alkynyl group). Herein, F¹ is a1,3-dipole when Q¹ is a dipolarophile and F¹ is a dipolarophile when Q¹is a 1,3-dipole, or F¹ is a diene when Q¹ is a dienophile and F¹ is adienophile when Q¹ is a diene. Most preferably, F¹ is a 1,3-dipole,preferably F¹ is or comprises an azido group.

Several examples of a functional group that is placed into a biomoleculeare shown in FIG. 2. FIG. 2 shows several structures of derivatives ofUDP sugars of galactosamine, which may be modified with e.g. athiopropionyl group (11a), an azidoacetyl group (11b), or anazidodifluoroacetyl group (11c) at the 2-position, or with an azidogroup at the 6-position of N-acetyl galactosamine (11d).

FIG. 3 schematically displays how any of the UDP-sugars 11a-d may beattached to a glycoprotein comprising a GlcNAc moiety 12 (e.g. amonoclonal antibody the glycan of which is trimmed by anendoglycosidase) under the action of a galactosyltransferase mutant or aGalNAc-transferase, thereby generating a 3-glycosidic 1-4 linkagebetween a GalNAc derivative and GlcNAc (compounds 13a-d, respectively).

Preferred examples of naturally present functional groups F¹ include athiol group and an amino group. Preferred examples of a functional groupthat is prepared by chemical synthesis for incorporation into thebiomolecule include a ketone group, a terminal alkyne group, an azidegroup, a cyclo(hetero)alkyne group, a cyclopropene group, or a tetrazinegroup.

As was described above, complementary reactive groups Q¹ and functionalgroups F¹ are known to a person skilled in the art, and several suitablecombinations of Q¹ and F¹ are described above, and shown in FIG. 6. Alist of complementary groups Q¹ and F¹ is disclosed in in Table 3.1,pages 230-232 of Chapter 3 of G. T. Hermanson, “BioconjugateTechniques”, Elsevier, 3^(rd) Ed. 2013 (ISBN: 978-0-12-382239-0), andthe content of this Table is expressly incorporated by reference herein.

An embodiment of the process according to the invention is depicted inFIG. 4. FIG. 4 shows how a modified antibody 13a-d may undergo abioconjugation process by means of nucleophilic addition with maleimide(as for 3-mercaptopropionyl-galactosamine-modified 13a leading tothioether conjugate 14, or for conjugation to an engineered cysteineresidue leading to thioether conjugate 17) or upon strain-promotedcycloaddition with a cyclooctyne reagent (as for 13b, 13c or 13d,leading to triazoles 15a, 15b or 16, respectively).

The invention further relates to a bioconjugate obtainable by theprocess according to the invention for the preparation of abioconjugate.

Use

In a fifth aspect, the invention concerns the use of the linkeraccording to the invention in bioconjugation for (a) improvingconjugation efficiency, (b) reducing aggregation, (c) increasingstability of a bioconjugate as defined herein and/or (d) increasingtherapeutic index of the bioconjugate. In one embodiment, the useaccording to the present aspect is for (a) improving conjugationefficiency in a bioconjugation reaction wherein the linker-conjugateaccording to the present invention reacts with a biomolecule, asdescribed herein. In one embodiment, the use according to the presentaspect is for (b) reducing aggregation during the bioconjugationreaction, e.g. the bioconjugation reaction according to the invention(in-process aggregation) and/or for reducing aggregation of thebioconjugate according to the invention (product aggregation). In oneembodiment, the use according to the present aspect is for (c)increasing stability of the bioconjugate according to the presentinvention, in particular for increasing stability towards hydrolysis. Inone embodiment, the use according to the present aspect is for (d)increasing therapeutic index of the bioconjugate.

A first advantages of the process for the preparation of a bioconjugateas described herein, and of the linker-conjugates and sulfamide linkeraccording to the invention is that conjugation efficiency increases incase a sulfamide linker is used instead of a typical polyethylene glycol(PEG) spacer. An additional advantage of a sulfamide group, inparticular of an acylsulfamide or a carbamoylsulfamide group, is itshigh polarity, which imparts a positive effect on the solubility of alinker comprising such group, and on the construct as a whole, before,during and after conjugation. In view of this increased polarity,conjugation with first precursor containing the sulfamide linkeraccording to the invention are particularly suited to conjugatehydrophobic target compounds to a biomolecule. The high polarity of thesulfamides also has a positive impact in case hydrophobic moieties areconjugated to a biomolecule of interest, which is known to require largeamounts of organic co-solvent during conjugation and/or induceaggregation of the bioconjugate. High levels of co-solvent (up to 50% ofDMA, DMF, or DMSO) may induce protein denaturation during theconjugation process and/or may require special equipment in themanufacturing process. Thus, the problem of aggregation associated withthe hydrophobic linking moieties in bioconjugates is efficiently solvedby using the sulfamide linker according to the invention in the spacerbetween the target molecule and the reactive group Q¹ in thelinker-conjugate in the formation of the bioconjugate. An additionaladvantage of a sulfamide linker according the invention, and its use inbioconjugation processes, is its ease of synthesis and high yields.Furthermore, the inventors have found that the bioconjugates accordingto the invention are more stable than bioconjugates not according to thepresent invention, i.e. not comprising the linker according to theinvention.

Lastly, the inventors surprisingly found that a bioconjugate accordingto the invention exhibits a greater therapeutic index compared to thesame bioconjugate, i.e. the same biomolecule, the same active substance(drug) and the same biomolecule drug ratio, but not containing a linkeraccording to the invention, i.e. wherein no sulfamide group (SG) ispresent. That the linker could have an effect on the therapeuticefficacy of a bioconjugate, such as an antibody-drug-conjugate, couldnot be envisioned based on the current knowledge. In the field, linkersare considered inert when it comes to treatment and are solely presentas a consequence of the preparation of the bioconjugate. That theselection of a specific linker has an effect on the therapeutic efficacyis unprecedented and a breakthrough discovery in the field ofbioconjugates, in particular antibody-drug-conjugates. The bioconjugatesaccording to the invention are thus more therapeutically effective asthe same bioconjugates, i.e. the same biomolecule, the same activesubstance (drug) and the same biomolecule-active substance ratio,containing a different linker. This finding has dramatic implications onthe treatment of subjects with the bioconjugate according to theinvention, as treatment doses may be lowered and as a consequencepotential, unwanted, side-effects are reduced. Alternatively, as aresult of the increased tolerability of the bioconjugate (i.e. increasedstability and low aggregation potential) according to the invention,treatment doses might be increased without the potential increase inunwanted side-effects.

The use according to the present aspect is largely non-medical. In oneembodiment, the use is a non-medical use for (a) improving conjugationefficiency, (b) reducing aggregation, (c) increasing stability of abioconjugate as defined herein and/or (d) increasing therapeutic indexof the bioconjugate.

The first aspect of the invention can also be worded as the linkeraccording to the invention for use in for (a) improving conjugationefficiency, (b) reducing aggregation, (c) increasing stability of abioconjugate as defined herein and/or (d) increasing therapeutic indexof the bioconjugate. In other words, the present aspect concerns the useof a linker according to the invention for (a) improving conjugationefficiency, (b) reducing aggregation, (c) increasing stability of abioconjugate as defined herein and/or (d) increasing therapeutic indexof the bioconjugate. The invention according to the present aspect canalso be worded as the use of a linker according to the invention in abioconjugate according to the invention, or in the preparation of abioconjugate according to the invention, for (a) improving conjugationefficiency, (b) reducing aggregation, (c) increasing stability of abioconjugate as defined herein and/or (d) increasing therapeutic indexof the bioconjugate. The use as defined herein may be referred to as anon-medical or non-therapeutic use.

The bioconjugate subject of the use according to the present aspect ispreferably obtainable by the process for the preparation of abioconjugate as defined above, more preferably the bioconjugate isobtained by the process for the preparation of a bioconjugate as definedabove. It was found that bioconjugates thus obtained had an even furtherimproved therapeutic index.

The inventors found that the linker according to the invention, ascomprised in the bioconjugates according to the invention, has an effecton both aspects of the therapeutic index: (a) on the therapeuticefficacy and (b) on the tolerability. Thus, the method for increasingthe therapeutic index is preferably for (a) increasing the therapeuticefficacy, and/or (b) increasing the tolerability of a bioconjugateaccording to the invention.

Thus, in one embodiment, the method according to the first aspect is forincreasing the therapeutic efficacy of a bioconjugate according to theinvention. Herein, “increasing the therapeutic efficacy” can also beworded as “lowering the effective dose” or “lowering the ED₅₀ value” or“increasing the protective index”. Likewise, in one embodiment, themethod according to the first aspect is for increasing the tolerabilityof a bioconjugate according to the invention. Herein, “increasing thetolerability” can also be worded as “increasing the maximum tolerateddose (MTD)”, “increasing the TD₅₀ value” or “reducing the toxicity”. Inone especially preferred embodiment, the method according to the firstaspect is for (a) increasing the therapeutic efficacy and (b) increasingthe tolerability of a bioconjugate according to the invention.

Medical Use

The invention thus concerns in a sixth aspect a method for the treatmentof a subject in need thereof, comprising the administration of thebioconjugate according to the invention as defined above. The subject inneed thereof is most preferably a cancer patient. The use ofbioconjugates, such as antibody-drug conjugates, is well-known in thefield of cancer treatment, and the bioconjugates according to theinvention are especially suited in this respect. The method as describedis typically suited for the treatment of cancer. In the method accordingto the sixth aspect, the bioconjugate is typically administered in atherapeutically effective dose. The bioconjugate according to theinvention is described a great detail above.

The sixth aspect of the invention can also be worded as a bioconjugateaccording to the invention for use in the treatment of a subject in needthereof, preferably for the treatment of cancer. In other words, thesecond aspect concerns the use of a bioconjugate according to theinvention for the preparation of a medicament pharmaceutical for use inthe treatment of a subject in need thereof, preferably for use in thetreatment of cancer.

Examples Examples 1-3: Preparation of Compound 25

The synthetic approach towards compound 25 is depicted here below.

Example 1: Preparation of Compound 21

To a solution of(R)-(+)-N-(tert-butoxycarbonyl)-O-(tert-butyldimethylsilyl)serinol (26;50 mg, 0.164 mmol) in DCM (1.6 mL) under a nitrogen atmosphere was addedchlorosulfonyl isocyanate (14.3 μL, 0.164 mmol) and the resultingmixture was stirred for 15 min. Then, Et₃N (68 μL, 0.491 mmol) wasadded, followed by benzylamine (18 μL, 0.164 mmol). After 1 h, DCM (10mL) and sat. NH₄Cl solution (10 mL) were added. The layers wereseparated and the water phase was extracted with DCM (2×10 mL) and EtOAc(3×10 mL). The combined organic layers were dried over sodium sulfate,filtered and concentrated in vacuo. Flash chromatography (10-50% EtOAcin pentane) afforded the product (72 mg, 0.14 mmol, 85%). LRMS (ESI+)calcd for C₂₂H₃₉N₃O₇SSi (M+H⁺) 518.24, found 518.28.

Example 2: Preparation of Compound 22

To a solution of compound 21 (72 mg, 0.14 mmol) in DCM (1 mL) was addedTFA (1 mL) and the reaction mixture was stirred at rt overnight. Thereaction was concentrated and co-evaporated with toluene (2×15 mL) toafford the crude product. 1H-NMR (400 MHz, CD₃OD): δ 7.38-7.24 (m, 5H),4.29-4.18 (m, 4H), 3.91-3.64 (m, 3H), 3.54-3.43 (m, 1H), 0.98-0.86 (m,5H), 0.15-0.04 (m, 5H). LRMS (ESI+) calcd for C₁₇H₃₁N₃O₅SSi (M+H+)418.18, found 418.20.

Example 3: Preparation of Compound 25

To a solution of the crude compound 22 (0.14 mmol) in MeCN (2 mL) undera nitrogen atmosphere, was added Et₃N (59 μL, 0.42 mmol), followed byaddition of compound 23 (79 mg, 0.21 mmol) in dry DMF (0.5 mL). Theresulting solution was stirred at rt overnight and concentrated toafford the crude product. LRMS (ESI+) calcd for C₃₂H₅₀N₄O₈SSi (M+H⁺)679.32, found 679.27.

The resulting compound 24 was dissolved in THF (1.5 mL) and TBAF (100mg) was added. The reaction was allowed to stir at rt for 24 h with anextra addition of TBAF (200 mg) after 18 h. After 24 h, the reaction wasconcentrated, taken up in EtOAc (15 mL) and washed with H₂O (2×10 mL).The water phase was extracted with DCM (20 mL), the combined organiclayers were dried over sodium sulfate, filtered and the solvent removedunder reduced pressure. Flash chromatography (80-100% EtOAc in pentane)afforded the product. ¹H-NMR (400 MHz, CDCl₃): δ 7.38-7.28 (m, 5H), 4.95(t, J=6 Hz, 1H), 4.28-4.20 (m, 4H), 4.17-4.08 (m, 3H), 3.82-3.65 (m,2H), 2.35-2.15 (m, 10H), 1.93-1.49 (m, 9H), 1.01-0.82 (m, 6H). LRMS(ESI+) calcd for C₂₆H₃₆N₄O₈S (M+H⁺) 565.23, found 565.25.

Examples 4-9: Preparation of Compound 32

The synthetic approach towards compound 32 is depicted here below.

Example 4: Preparation of Compound 27

To a solution of compound 26 (200 mg, 0.65 mmol) in DCM (6.5 mL) under anitrogen atmosphere was added chlorosulfonyl isocyanate (CSI, 63 μL,0.72 mmol) and the resulting mixture was stirred for 30 min, when TLCshowed consumption of the starting material. Then, Et₃N (270 μL, 1.95mmol) was added followed by 2-(2-methoxyethoxy)ethanamine (83 μL, 0.65mmol). After 45 min, DCM (15 mL) and sat. NH₄Cl solution (10 mL) wereadded. The layers were separated and the water phase was extracted withEtOAc (3×10 mL). The combined organic layers were dried over sodiumsulfate, filtered and concentrated in vacuo. Flash chromatography(20-70% EtOAc in pentane) afforded the product (287 mg, 0.54 mmol, 73%).¹H-NMR (400 MHz, CDCl₃): δ 6.16 (br s, 1H), 5.04 (d, J=8.8 Hz, 1H), 4.29(dd, J=11.2, 5.6 Hz, 1H), 4.22-4.15 (m, 1H), 3.95 (br s, 1H), 3.70 (dd,J=10, 3.2 Hz, 1H), 3.65-3.60 (m, 5H), 3.59-3.55 (m, 2H), 3.41 (s, 3H),3.34 (br s, 2H), 1.45 (s, 9H), 0.89 (s, 9H), 0.06 (s, 6H). LRMS (ESI+)calcd for C₂₀H₄₃N₃O₉SSi (M+H⁺) 530.26, found 530.21.

Example 5: Preparation of Compound 28

To a solution of compound 27 (287 mg, 0.54 mmol) in DCM (4 mL) was addedTFA (1 mL) and the reaction mixture was stirred at rt for 1.5 h. Thereaction was concentrated and co-evaporated with toluene (2×30 mL) toafford the crude product. LRMS (ESI+) calcd for C₁₅H₃₅N₃O₇SSi (M+H⁺)430.20, found 430.20.

Example 6: Preparation of Compound 29

To a solution of the crude amine (0.27 mmol) in DCM (2 mL) under anitrogen atmosphere was added Et₃N (149 μL, 1.1 mmol), followed byaddition of compound 23 (122 mg, 0.32 mmol) in DCM (1 mL). The resultingsolution was stirred at rt overnight and analysed by mass spectrometry.All starting material was consumed, the coupled product and productcontaining the free alcohol were formed. The reaction mixture was usedneat for flash chromatography (50-100% EtOAc in pentane, then 1-10% MeOHin EtOAc) to afford both observed products, OTBDMS derivative 29 (69 mg,0.09 mmol, 37%) and OH derivative 30 (113 mg, 0.18 mmol, 65%). For 29:¹H-NMR (400 MHz, CDCl₃): δ 5.1 (br s, 1H), 4.32 (d, J=8.8 Hz, 2H),4.23-4.11 (m, 3H), 3.70 (dd, J=10.4, 3.2 Hz, 1H), 3.66-3.58 (m, 8H),3.57-3.51 (m, 2H), 3.40-3.34 (m, 4H), 3.30 (t, J=4.4 Hz, 2H), 3.24-3.18(m, 2H), 2.34-2.15 (m, 8H), 1.90-1.73 (m, 2H), 1.64-1.50 (m, 2H),1.42-1.28 (m, 2H), 0.99-0.80 (m, 11H), 0.06 (s, 6H). LRMS (ESI+) calcdfor C₃₀H₅₄N₄O₁₀SSi (M+H⁺) 691.34, found 691.35. For 30: LRMS (ESI+)calcd for C₂₄H₄₀N₄O₁₀S (M+H⁺) 577.25, found 577.25.

Example 7: Preparation of Compound 30

To a solution of the protected alcohol 29 (69 mg, 0.099 mmol) in dry THF(1 mL) was added TBAF (120 μL, 1 M solution in THF) under a nitrogenatmosphere. The reaction was stirred at rt and the conversion monitoredby mass spectrometry. After 3 h, complete conversion was observed andthe reaction was concentrated to afford the crude product. LRMS (ESI+)calcd for C₂₄H₄₀N₄O₁₀S (M+H⁺) 577.25, found 577.25.

Example 8: Preparation of Compound 31

To a solution of compound 30 (55 mg, 0.095 mmol) in DCM/DMF 9/1 (1 mL)were added DSC (20 mg, 0.076 mmol) and Et₃N (40 μL, 0.286 mmol). Thereaction mixture was stirred at rt and the conversion monitored by massspectrometry. After 3 h, 100 μL was taken out for the follow-up reaction(coupling to VC-PAB-MMAE). The remaining 900 μL was used neat for flashchromatography (0-10% MeOH in EtOAc) to afford the product (34 mg, 0.047mmol, 50%). LRMS (ESI+) calcd for C₂₉H₄₃N₅O₁₄S (M+H⁺) 718.26, found718.35.

Example 9: Preparation of Compound 32

Val-Cit-PABC-MMAE (17 mg, 13 μmol) was dissolved in DMF (500 μL) andEt₃N (2.1 μL, 15 μmol) was added. Then, 100 μL of the reaction mixtureof the DSC activation containing approx. 5 μmol activated alcohol, wasadded to the reaction and the mixture was allowed to stand overnight atrt. 2,2′-(ethylenedioxy)bis(ethylamine) (2.2 μL, 15 μmol) was added toquench possible remaining activated reagent. After 2 h, the resultingsolution was purified by preparative HPLC (70:30-10:90 H₂O:MeCN+1% AcOH)to afford the product (3.8 mg, 2.2 μmol, approx. 44%). LRMS (ESI+) calcdfor C₈₃H₁₃₂N₁₄O₂₃S (M+H⁺) 1727.11, found 1727.09.

Examples 10-14: EndoSH

In one aspect, the invention concerns a fusion enzyme comprising twoendoglycosidases. In a particular example the two endoglycosidases EndoSand EndoH are connected via a linker, preferably a-(Gly₄Ser)₃-(His)₆-(Gly₄Ser)₃-linker. The fusion enzyme according to theinvention as also referred to as EndoSH. The enzyme according to theinvention has at least 50% sequence identity with SEQ ID NO: 1,preferably at least 70%, more preferably at least 80% sequence identitywith SEQ ID NO: 1, such as at least 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequenceidentity with SEQ ID NO: 1.

Identity can be readily calculated by known methods and/or computerprogram methods known in the art such as BLASTP publicly available fromNCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIHBethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410(1990). Preferably, the enzyme of the invention, having the aboveindicated sequence identity to SEQ ID NO: 1, has EndoS and EndoHactivity. Most preferably, the enzyme according to the invention has100% sequence identity with SEQ ID NO: 1.

Also encompassed are fusion enzymes of EndoS and EndoH, wherein thelinker is replaced by another suitable linker known in the art, whereinsaid linker may be a rigid, or flexible. Preferably, said linker is aflexible linker allowing the adjacent protein domains to move relativefreely to one another. Preferably, said flexible linker is composed ofamino residues like glycine, serine, histidine and/or alanine and has alength of 3 to 59 amino acid residues, preferably 10 to 45 or 15 to 40amino acid residues, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 amino acidresidues, or 20 to 38, 25 to 37 or 30 to 36 amino acid residues.Optionally, the fusion enzyme is covalently linked to, or comprises, atag for ease of purification and or detection as known in the art, suchas an Fc-tag, FLAG-tag, poly(His)-tag, HA-tag and Myc-tag.

Trimming of glycoproteins is known in the art, from e.g. WO 2007/133855or WO 2014/065661. The enzyme according to the invention exhibits bothEndoS and EndoH activity, and is capable of trimming glycans onglycoproteins (such as antibodies) at the core GlcNAc unit, leaving onlythe core GlcNAc residue on the glycoprotein (EndoS activity) as well aswell as splitting off high-manose glycans (EndoH activity).Surprisingly, both activities of the fusion enzyme function smoothly ata pH around 7-8, while monomeric EndoH requires a pH of 6 to operateoptimally. The fusion enzyme according to the invention can be preparedby routine techniques in the art, such as introducing an expressionvector (e.g. plasmid) comprising the enzyme coding sequence into a hostcell (e.g. E. coli) for expression, from which the enzyme can beisolated. A possible approach for the preparation and purification ofthe fusion enzyme according to the invention is given in examples 10-12,and its functioning is demonstrated in examples 13 and 14, whereintrastuzumab and high-mannose trastuzumab are efficiently trimmed in asingle step. Another example of the efficient trimming of glycans by thefusion protein EndoSH is provided in example 17.

Example 10: Cloning of Fusion Protein EndoSH into (pET22B) ExpressionVector

A pET22B-vector containing an EndoS-(G₄S)₃-(His)₆-(G₄S)₃-EndoH (EndoSH)coding sequence (EndoSH being identified by SEQ ID NO: 1) betweenEcoRI-Hindlll sites was obtained from Genscript. The DNA sequence forthe EndoSH fusion protein consists of the encoding residues 48-995 ofEndoS fused via an N-terminal linked glycine-serine (GS) linker toEndoH. The glycine-serine (GS) linker comprises a-(G₄S)₃-(His)₆-(G₄S)₃-format, allowing spacing of the two enzymes and atthe same time introducing a IMAC-purification tag.

Example 11: E. coli Expression of Fusion Protein EndoSH

Expression of the EndoSH fusion protein (identified by SEQ ID NO: 1)starts with the transformation of the plasmid (pET22b-EndoSH) into BL21cells. Next step is the inoculation of 500 mL culture (LBmedium+Ampilicin) with BL21 cells. When the OD600 reached 0.7 thecultures were induced with 1 mM IPTG (500 μL of 1M stock solution).

Example 12: Purification of Fusion Protein EndoSH from E. coli

After overnight induction at 37° C. the cultures were pelleted bycentrifugation. The pellets were resuspended in 40 mL PBS and incubatedon ice with 5 ml lysozyme (10 mg/mL) for 30 minutes. After half an hour5 ml 10% Triton-X-100 was added and sonicated (10 minutes) on ice. Afterthe sonification the cell debris was removed by centrifugation (10minutes 8000×g) followed by filtration through a 0.22 μM-pore diameterfilter. The soluble extract was loaded onto a HisTrap HP 5 mL column (GEHealthcare). The column was first washed with buffer A (20 mM Trisbuffer, 20 mM imidazole, 500 mM NaCl, pH 7.5). Retained protein waseluted with buffer B (20 mM Tris, 500 mM NaCl, 250 mM imidazole, pH 7.5,10 mL). Fractions were analyzed by SDS-PAGE on polyacrylamide gels(12%). The fractions that contained purified target protein werecombined and the buffer was exchanged against 20 mM Tris pH 7.5 and 150mM NaCl by dialysis performed overnight at 4° C. The purified proteinwas concentrated to at least 2 mg/mL using an Amicon Ultra-0.5,Ultracel-10 Membrane (Millipore). The product is stored at −80° C. priorto further use.

Example 13: Trimming of Trastuzumab by EndoSH

Trastuzumab (obtained from Epirus biopharma (Utrecht, The Netherlands))in 25 mM Tris buffer pH 8) (14 mg/mL) was trimmed using a concentrationof either 0.1 or 1 w/w % EndoSH. The reactions, 350 μg trastuzumab (25μL) and the appropriate amount of EndoSH, were stirred at 37° C. andanalyzed by MS analysis over time, 1 to 3 hours. Samples were subjectedto Fabricator treatment prior to analysis. Full conversions to thetrimmed product, which trimmed to the core GlcNAc sugar residue, wasobserved after 1 hour at 37° C. with 0.1 w/w % EndoSH.

Example 14: Trimming of High-Mannose Trastuzumab by Fusion ProteinEndoSH

Trastuzumab having high-mannose glycans (obtained via transientexpression in CHO K1 cells in the presence of kifunensine performed byEvitria (Zurich, Switzerland)) (14 mg/mL) in 25 mM Tris buffer pH 8, wastrimmed using a concentration of either 0.1 or 1 w/w % EndoSH. Thereactions, 350 μg high-mannose trastuzumab (25 μL) and the appropriateamount of EndoSH, were stirred at 37° C. and analyzed by MS analysisover time, 1-3 hours. Samples were subjected to Fabricator treatmentprior to analysis. Full conversions to the trimmed product, whichtrimmed to the core GlcNAc sugar residue, was observed after 3 hours at37° C. with 1 w/w % EndoSH.

RP-HPLC Analysis of Reduced Monoclonal Antibodies

Prior to RP-HPLC analysis samples were reduced by incubating a solutionof 10 μg (modified) IgG for 15 minutes at 37° C. with 10 mM DTT and 100mM Tris pH 8.0 in a total volume of 50 μL. A solution of 49% ACN, 49% MQand 2% formic acid (50 μL) was added to the reduced sample. Reversephase HPLC was performed on a Agilent 1100 HPLC using a ZORBAXPhoroshell 300SB-C8 1×75 5 μm (Agilent Technologies) column run at 1ml/min at 70° C. using a 16.9 minute linear gradient from 25 to 50%buffer B (with buffer A=90% MQ, 10% ACN, 0.1% TFA and buffer B=90% ACN,10% MQ, 0.1% TFA).

Mass Spectral Analysis of Monoclonal Antibodies

Prior to mass spectral analysis, IgGs were either treated with DTT,which allows analysis of both light and heavy chain, or treated withFabricator™ (commercially available from Genovis, Lund, Sweden), whichallows analysis of the Fc/2 fragment. For analysis of both light andheavy chain, a solution of 20 μg (modified) IgG was incubated for 5minutes at 37° C. with 100 mM DTT in a total volume of 4 μL. If present,azide-functionalities are reduced to amines under these conditions. Foranalysis of the Fc/2 fragment, a solution of 20 μg (modified) IgG wasincubated for 1 hour at 37° C. with Fabricator™ (1.25 U/μL) inphosphate-buffered saline (PBS) pH 6.6 in a total volume of 10 μL. Afterreduction or Fabricator-digestion the samples were washed trice withmilliQ using an Amicon Ultra-0.5, Ultracel-10 Membrane (Millipore)resulting in a final sample volume of approximately 40 μL. Next, thesamples were analyzed by electrospray ionization time-of-flight(ESI-TOF) on a JEOL AccuTOF. Deconvoluted spectra were obtained usingMagtran software

Examples 15-19: Preparation of cAC10 Bioconjucate

The preparation of modified biomolecule 13d, is performed according tothe procedure as is described below utilizing an endoglycosidase fusionprotein EndoS-linker-EndoH (also referred to as EndoSH, identified bySEQ ID NO: 1) for trimming of the glycans of cAC10. In the second stepthe trimmed cAC10 was converted to the azido-modified mAb 13d throughthe action of His-TnGalNAcT in the presence of 6-N₃-GalNAc-UDP(commercially available from GlycoHub) as a substrate.

Example 15: Transient Expression and Purification of cAC10

cAC10 was transiently expressed in CHO K1 cells by Evitria (Zurich,Switzerland) at 5 L scale. The supernatant was purified using a XK 26/20column packed with 50 mL protein A sepharose. In a single run 5 Lsupernatant was loaded onto the column followed by washing with at least10 column volumes of 25 mM Tris pH 7.5, 150 mM NaCl. Retained proteinwas eluted with 0.1 M Glycine pH 2.7. The eluted cAC10 was immediatelyneutralized with 1.5 M Tris-HCl pH 8.8 and dialyzed against 25 mM TrispH 8.0. Next the IgG was concentrated to approximately 20 mg/mL using aVivaspin Turbo 15 ultrafiltration unit (Sartorius) and stored at −80° C.prior to further use.

Example 16: Transient Expression and Purification ofhis-TnGalNAcT(33-421)

His-TnGalNAcT(33-421) (identified by SEQ ID NO: 2) was transientlyexpressed in CHO K1 cells by Evitria (Zurich, Switzerland) at 5 L scale.The supernatant was purified using a XK 16/20 column packed with 25 mLNi sepharose excel (GE Healthcare). Each run approximately 1.5 Lsupernatant was loaded onto the column followed by washing with at least10 column volumes of buffer A (20 mM Tris buffer, 5 mM imidazole, 500 mMNaCl, pH 7.5). Retained protein was eluted with buffer B (20 mM Tris,500 mM NaCl, 500 mM imidazole, pH 7.5). The buffer of the elutedfractions was exchanged to 25 mM Tris pH 8.0 using a HiPrep H26/10desalting column (GE Healthcare). The purified protein was concentratedto at least 3 mg/mL using a Vivaspin Turbo 4 ultrafiltration unit(Sartorius) and stored at −80° C. prior to further use.

Example 17: Preparation of Trimmed cAC10 by Means of Fusion ProteinEndoSH

Glycan trimming of cAC10 (obtained via transient expression in CHO K1cells performed by Evitria (Zurich, Switzerland) was performed withfusion protein EndoSH. Thus, cAC10 (14.5 mg/mL) was incubated withEndoSH (1 w/w %) in 25 mM Tris pH 7.5 with 150 mM NaCl for approximately16 hours at 37° C. The trimmed IgG was dialyzed against 3×1 L of 25 mMTris-HCl pH 8.0. Mass spectral analysis of a fabricator-digested sampleshowed three peaks of the Fc/2-fragment belonging to one major product(observed mass 24105 Da, approximately 80% of total Fc/2 fragment),corresponding to core GlcNAc(Fuc)-substituted cAC10, and two minorproducts (observed masses of 23959 and 24233 Da, approximately 5 and 15%of total Fc/2 fragment), corresponding to core GlcNAc-substituted cAC10and core GlcNAc(Fuc)-substituted cAC10 with C-terminal lysine.

Example 18: Glycosyltransfer of the 6-N₃-GalNAc-UDP to Trimmed cAC10Under the Action of TnGalNAcT

Substrate 6-N₃-GalNAc-UDP (11d) is used for the preparation of themodified biomolecule cAC10-(6-N₃-GalNAc)₂ 13d, suitable as biomoleculein the context of the invention. Trimmed cAC10 (10 mg/mL), obtained byEndoSH treatment of cAC10 as described above in example 17, wasincubated with the substrate 6-N₃-GalNAc-UDP (2.5 mM, commerciallyavailable from GlycoHub) and 0.5 mg/mL His-TnGalNAcT(33-421) (5 w/w %)in 10 mM MnCl2 and 25 mM Tris-HCl pH 8.0 at 30° C. After 3 hours theamount of His-TnGalNAcT(33-421) was increased to a final concentrationof 1 mg/mL (10 w/w %) and the reaction was incubated overnight at 30° C.Biomolecule 13d was purified from the reaction mixture on a HiTrapMabSelect SuRe 5 ml column (GE Healthcare) using an AKTA purifier-10 (GEHealthcare). The eluted IgG was immediately neutralized with 1.5 MTris-HCl pH 8.8 and dialyzed against PBS pH 7.4. Next the IgG wasconcentrated using an Amicon Ultra-0.5, Ultracel-10 Membrane (Millipore)to a concentration of 23.38 mg/mL. Mass spectral analysis of afabricator-digested sample showed three peaks of the Fc/2-fragmentbelonging to one major product (observed mass 24333 Da, approximately80% of total Fc/2 fragment), corresponding to core6-N₃-GalNAc-GlcNAc(Fuc)-substituted cAC10, and two minor products(observed masses of 24187 and 24461 Da, approximately 5 and 15% of totalFc/2 fragment), corresponding to core 6-N₃-GalNAc-GlcNAc-substitutedcAC10 and core 6-N₃-GalNAc-GlcNAc(Fuc)-substituted cAC10 with C-terminallysine.

Example 19: Conjugation of 13d with 32 to Obtain Conjugate 33

The bioconjugate according to the invention was prepared according tothe following scheme, by conjugation of compound 32 to modifiedbiomolecule 13d:

To a solution of cAC(azide)₂ (13d) (287 μL, 6.7 mg, 23.38 mg/ml in PBSpH 7.4) was added compound 32 (90 μL, 10 mM solution in DMF). Thereaction was incubated at rt overnight followed by purification on aSuperdex200 10/300 GL (GE Healthcare) on an AKTA Purifier-10 (GEHealthcare). Mass spectral analysis of the fabricator-digested sampleshowed one major product (observed mass 26092 Da, approximately 90% oftotal Fc/2 fragment), corresponding to the conjugated Fc/2 fragment.RP-HPLC analysis of the reduced sample indicated an average DAR of 1.81.

Sequence identification of fusion protein EndoSH (SEQ. ID NO: 1):    1MPSIDSLHYL SENSKKEFKE ELSKAGQESQ KVKEILAKAQ QADKQAQELA   51KMKIPEKIPM KPLHGPLYGG YFRTWHDKTS DPTEKDKVNS MGELPKEVDL  101AFIFHDWTKD YSLFWKELAT KHVPKLNKQG TRVIRTIPWR FLAGGDNSGI  151AEDTSKYPNT PEGNKALAKA IVDEYVYKYN LDGLDVDVEH DSIPKVDKKE  201DTAGVERSIQ VFEEIGKLIG PKGVDKSRLF IMDSTYMADK NPLIERGAPY  251INLLLVQVYG SQGEKGGWEP VSNRPEKTME ERWQGYSKYI RPEQYMIGFS  301FYEENAQEGN LWYDINSRKD EDKANGINTD ITGTRAERYA RWQPKTGGVK  351GGIFSYAIDR DGVAHQPKKY AKQKEFKDAT DNIFHSDYSV SKALKTVMLK  401DKSYDLIDEK DFPDKALREA VMAQVGTRKG DLERFNGTLR LDNPAIQSLE  451GLNKFKKLAQ LDLIGLSRIT KLDRSVLPAN MKPGKDTLET VLETYKKDNK  501EEPATIPPVS LKVSGLTGLK ELDLSGFDRE TLAGLDAATL TSLEKVDISG  551NKLDLAPGTE NRQIFDTMLS TISNHVGSNE QTVKFDKQKP TGHYPDTYGK  601TSLRLPVANE KVDLQSQLLF GTVTNQGTLI NSEADYKAYQ NHKIAGRSFV  651DSNYHYNNFK VSYENYTVKV TDSTLGTTTD KTLATDKEET YKVDFFSPAD  701KTKAVHTAKV IVGDEKTMMV NLAEGATVIG GSADPVNARK VFDGQLGSET  751DNISLGWDSK QSIIFKLKED GLIKHWRFFN DSARNPETTN KPIQEASLQI  801FNIKDYNLDN LLENPNKFDD EKYWITVDTY SAQGERATAF SNTLNNITSK  851YWRVVFDTKG DRYSSPVVPE LQILGYPLPN ADTIMKTVTT AKELSQQKDK  901FSQKMLDELK IKEMALETSL NSKIFDVTAI NANAGVLKDC IEKRQLLKKG  951GGGSGGGGSG GGGSHHHHHH EFGGGGSGGG GSGGGGS APA PVKQGPTSVA 1001YVEVNNNSML NVGKYTLADG GGNAFDVAVI FAANINYDTG TKTAYLHFNE 1051NVQRVLDNAV TQIRPLQQQG IKVLLSVLGN HQGAGFANFP SQQAASAFAK 1101QLSDAVAKYG LDGVDFDDEY AEYGNNGTAQ PNDSSFVHLV TALRANMPDK 1151IISLYNIGPA ASRLSYGGVD VSDKFDYAWN PYYGTWQVPG IALPKAQLSP 1201AAVEIGRTSR STVADLARRT VDEGYGVYLT YNLDGGDRTA DVSAFTRELY 1251 GSEAVRTP(linker is underlined, EndoH sequence is denoted in italics)Sequence of His-TnGalNAcT(33-421) (SEQ. ID NO: 2):    1MGSSHHHHHH SSGLVPRGSH MSPLRTYLYT PLYNATQPTL RNVERLAANW PKKIPSNYIE   61DSEEYSIKNI SLSNHTTRAS VVHPPSSITE TASKLDKNMT IQDGAFAMIS PTPLLITKLM  121DSIKSYVTTE DGVKKAEAVV TLPLCDSMPP DLGPITLNKT ELELEWVEKK FPEVEWGGRY  181SPPNCTARHR VAIIVPYRDR QQHLAIFLNH MHPFLMKQQI EYGIFIVEQE GNKDFNRAKL  241MNVGFVESQK LVAEGWQCFV FHDIDLLPLD TRNLYSCPRQ PRHMSASIDK LHFKLPYEDI  301FGGVSAMTLE QFTRVNGFSN KYWGWGGEDD DMSYRLKKIN YHIARYKMSI ARYAMLDHKK  361STPNPKRYQL LSQTSKTFQK DGLSTLEYEL VQVVQYHLYT HILVNIDERS

The invention claimed is:
 1. A compound comprising a target molecule Dcovalently connected to a reactive group Q¹ via a linker represented bythe following formula:

wherein: BM is a branching moiety; E is a capping group; SG is asulfamide group according to formula (1); b is independently 0 or 1; cis 0 or 1; d is 0 or 1; e is 0 or 1; f is an integer in the range of 1to 10; g is 0 or 1; i is 0 or 1; k is 0 or 1; l is 0 or 1; Sp¹ is aspacer moiety; Sp² is a spacer moiety; Sp³ is a spacer moiety; Sp⁴ is aspacer moiety; Sp⁵ is a spacer moiety; Sp⁶ is a spacer moiety; Z¹ is aconnecting group; Z² is a connecting group, wherein one of the bondslabelled with * is connected to reactive group Q¹ and one of the bondslabelled with * is connected to target molecule D, and wherein thesulfamide group SG is represented by formula (1):

wherein a is 0 or 1; and R¹ is selected from the group consisting ofhydrogen, C¹- C²⁴ alkyl groups, C³- C²⁴ cycloalkyl groups, C²- C²⁴(hetero)aryl groups, C³- C²⁴ alkyl(hetero)aryl groups and C₃- C₂₄(hetero)arylalkyl groups, wherein the C₁- C₂₄ alkyl groups, C₃- C₂₄cycloalkyl groups, C₂- C₂₄ (hetero)aryl groups, C₃- C₂₄alkyl(hetero)aryl groups and C₃- C₂₄ (hetero)arylalkyl groups areoptionally substituted and optionally interrupted by one or moreheteroatoms selected from O, S and NR³ wherein R³ is independentlyselected from the group consisting of hydrogen and C₁- C₄ alkyl groups,or R¹ is a further target molecule D, wherein the target molecule isoptionally connected to N via a spacer moiety, and wherein one of thebonds labelled with * is connected to the branching moiety, optionallyvia spacer Sp⁵, and the other bond labelled with * to a capping group E,optionally via spacer Sp⁶.
 2. The compound according to claim 1, whereinthe capping moiety E is selected from hydrogen, C₁- C₂₄ alkyl groups,C₃- C₂₄ cycloalkyl groups, C₂- C₂₄ (hetero)aryl groups, C₃- C₂₄alkyl(hetero)aryl groups, C₃- C₂₄ (hetero)arylalkyl groups, wherein theC₁- C₂₄ alkyl groups, C₃- C₂₄ cycloalkyl groups, C₂- C₂₄ (hetero)arylgroups, C₃- C₂₄ alkyl(hetero)aryl groups and C₃- C₂₄ (hetero)arylalkylgroups are optionally substituted and optionally interrupted by one ormore heteroatoms selected from O, S and NR³ wherein R³ is independentlyselected from the group consisting of hydrogen and C₁- C₄ alkyl groups,or E is a further target moiety D.
 3. The compound according to claim 2,wherein capping moiety E is selected from a polyethylene glycol grouprepresented by —(CH₂ CH₂ O)sCH₃, wherein s is an integer in the range of1- 10, a C₁- C₂₄ alkyl group, C₂- C₂₄ (hetero)aryl group or a C₃- C₂₄alkyl(hetero)aryl group.
 4. The compound according to claim 3, whereincapping moiety E is a polyethylene glycol group represented by —(CH₂CH₂O)sCH₃, wherein s is an integer in the range of 2- 4, or a benzylgroup.
 5. The compound according to claim 1, wherein branching moiety BMis selected from a carbon atom, a nitrogen atom, a phosphorus atom, anaromatic ring, a (hetero)cycle or a polycyclic moiety.
 6. The compoundaccording to claim 1, wherein Sp¹, Sp², Sp³ and Sp⁴, if present, areindependently selected from the group consisting of linear or branchedC₁-C ₂₀ alkylene groups, the alkylene groups being optionallysubstituted and optionally interrupted by one or more heteroatomsselected from the group consisting of O, S and N11 ³, wherein R³ isindependently selected from the group consisting of hydrogen and C₁-C₄alkyl groups, and wherein Q¹ is according to formula (9a), (9q), (9n),(9o) or (9p):

wherein U is 0 or NR⁹, and R⁹ is hydrogen, a linear or branched C₁- C₁₂alkyl group or a C₄- C₁₂ (hetero)aryl group.
 7. A compound comprising atarget molecule D covalently connected to a biomolecule B via a linkerrepresented by the following formula:

wherein: BM is a branching moiety; E is a capping group; SG is asulfamide group according to formula (1); b is independently 0 or 1; cis 0 or 1; d is 0 or 1; e is 0 or 1; f is an integer in the range of 1to 10; g is 0 or 1; i is 0 or 1; k is 0 or 1; l is 0 or 1; Sp¹ is aspacer moiety; Sp² is a spacer moiety; Sp^(a) is a spacer moiety; Sp⁴ isa spacer moiety; Sp⁵ is a spacer moiety; Sp⁶ is a spacer moiety; Z¹ is aconnecting group; Z² is a connecting group, wherein one of the bondslabelled with * is connected to biomolecule B and one of the bondslabelled with * is connected to target molecule D, and wherein thesulfamide group SG is represented by formula (1):

wherein a is 0 or 1; and R¹ is selected from the group consisting ofhydrogen, C₁- C₂₄ alkyl groups, C₃- C₂₄ cycloalkyl groups, C₂- C₂₄(hetero)aryl groups, C₃- C₂₄ alkyl(hetero)aryl groups and C₃- C₂₄(hetero)arylalkyl groups, wherein the C₁- C₂₄ alkyl groups, C₃- C₂₄cycloalkyl groups, C₂- C₂₄ (hetero)aryl groups, C₃- C₂₄alkyl(hetero)aryl groups and C₃- C₂₄ (hetero)arylalkyl groups areoptionally substituted and optionally interrupted by one or moreheteroatoms selected from O, S and NR³ wherein R³ is independentlyselected from the group consisting of hydrogen and C₁- C₄ alkyl groups,or R¹ is a further target molecule D, wherein the target molecule isoptionally connected to N via a spacer moiety, and wherein one of thebonds labelled with * is connected to the branching moiety, optionallyvia spacer Sp⁵, and the other bond labelled with * to a capping group E,optionally via spacer Sp⁶.
 8. The compound according to claim 7, whereinthe capping moiety E is selected from hydrogen, C₁- C₂₄ alkyl groups,C₃- C₂₄ cycloalkyl groups, C₂- C₂₄ (hetero)aryl groups, C₃- C₂₄alkyl(hetero)aryl groups, C₃- C₂₄ (hetero)arylalkyl groups, wherein theC₁- C₂₄ alkyl groups, C₃- C₂₄ cycloalkyl groups, C₂- C₂₄ (hetero)arylgroups, C₃- C₂₄ alkyl(hetero)aryl groups and C₃-C₂₄ (hetero)arylalkylgroups are optionally substituted and optionally interrupted by one ormore heteroatoms selected from 0, S and NR³ wherein R³ is independentlyselected from the group consisting of hydrogen and C₁- C₄ alkyl groups,or E is a further target moiety D.
 9. The compound according to claim 7,wherein branching moiety BM is selected from a carbon atom, a nitrogenatom, a phosphorus atom, an aromatic ring, a (hetero)cycle or apolycyclic moiety.
 10. A process for the preparation of a bioconjugate,comprising reacting a reactive group Q¹ of the compound according toclaim 1 with a functional group F¹ of a biomolecule (B).
 11. The processaccording to claim 10, wherein the reaction is a cycloaddition or aMichael reaction.
 12. The process according to claim 10, wherein thereaction is a Diels-Alder reaction or a 1,3-dipolar cycloaddition.
 13. Amethod for: (a) improving conjugation efficiency in the preparation ofthe bioconjugate, (b) reducing aggregation during the preparation of thebioconjugate and/or of the bioconjugate, (c) increasing stability of thebioconjugate, and/or (d) increasing therapeutic index of thebioconjugate, the method comprising conjugating a biomolecule (B) to alinker represented by the following formula:

wherein: BM is a branching moiety; E is a capping group; SG is asulfamide group according to formula (1); b is independently 0 or 1; cis 0 or 1; d is 0 or 1; e is 0 or 1; f is an integer in the range of 1to 10; g is 0 or 1; i is 0 or 1; k is 0 or 1; l is 0 or 1; Sp¹ is aspacer moiety; Sp² is a spacer moiety; Sp³ is a spacer moiety; Sp⁴ is aspacer moiety; Sp⁵ is a spacer moiety; Sp⁶ is a spacer moiety; Z′ is aconnecting group; Z² is a connecting group, wherein one of the bondslabelled with * is connected to biomolecule B and one of the bondslabelled with * is connected to target molecule D, and wherein thesulfamide group SG is represented by formula (1):

wherein a is 0 or 1; and R¹ is selected from the group consisting ofhydrogen, C₁- C₂₄ alkyl groups, C₃- C₂₄ cycloalkyl groups, C₂- C₂₄(hetero)aryl groups, C₃- C₂₄ alkyl(hetero)aryl groups and C₃- C₂₄(hetero)arylalkyl groups, wherein the C₁- C₂₄ alkyl groups, C₃- C₂₄cycloalkyl groups, C₂- C₂₄ (hetero)aryl groups, C₃- C₂₄alkyl(hetero)aryl groups and C₃- C₂₄ (hetero)arylalkyl groups areoptionally substituted and optionally interrupted by one or moreheteroatoms selected from O, S and NR³ wherein R³ is independentlyselected from the group consisting of hydrogen and C₁- C₄ alkyl groups,or R¹ is a further target molecule D, wherein the target molecule isoptionally connected to N via a spacer moiety, and wherein one of thebonds labelled with * is connected to the branching moiety, optionallyvia spacer Sp⁵, and the other bond labelled with * to a capping group E,optionally via spacer Sp⁶.
 14. A method of treatment comprisingadministering an effective amount of a compound according to claim 7 toa subject suffering from cancer.